Cationic lipids for therapeutic agent delivery formulations

ABSTRACT

Here described are compounds of formula I: 
                         
wherein R 1  and R 2  is independently selected from a group consisting of C 10  to C 18  alkyl, C 12  to C 18  alkenyl, and oleyl group; wherein R 3  and R 4  are independently selected from a group consisting of C 1  to C 6  alkyl, and C 2  to C 6  alkanol; wherein X is selected from a group consisting of —CH 2 —, —S—, and —O— or absent; wherein Y is selected from —(CH 2 ) n , —S(CH 2 ) n , —O(CH 2 ) n —, thiophene, —SO 2 (CH 2 ) n —, and ester, wherein n=1-4; wherein a=1-4; wherein b=1-4; wherein c=1-4; and wherein Z is a counterion; as well as compositions and pharmaceutical formulations including compounds of formula I which are useful for the delivery of therapeutic agents; and methods of using these compositions and formulations.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.13/492,650, filed Jun. 8, 2012, which claims the benefit of U.S.Provisional Application No. 61/494,710, filed Jun. 8, 2011, which areherein incorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 17, 2014, isnamed Sequence_Listing_CRF_(—)101025_(—)000052 and is 4,854 bytes insize.

TECHNICAL FIELD

The present invention is directed to the use of cationic lipids forenhancing the delivery of therapeutic agents.

BACKGROUND

A number of techniques are available for delivering a therapeutic agentsuch as siRNA into a cell, including the use of viral transfectionsystems and non-viral transfection systems. Non-viral transfectionsystems can include, for example, polymers, lipids, liposomes, micelles,dendrimers, and nanomaterials. Examples of polymers that have previouslybeen studied for cell transfection include cationic polymers such aspoly(L-lysine) (PLL), polyethyleneimine (PEI), chitosan, andpoly(2-dimethylamino)ethyl methacrylate (pDMAEMA).

Each type of system has its respective advantages and drawbacks. Forexample, viral systems can yield high transfection efficiency, but maynot be as safe as some non-viral systems. In addition, viral systems canbe complicated and/or expensive to prepare. Non-viral transfectionsystems, such as cationic polymers, have been reported to transferplasmid DNA into cells. However, some drawbacks to the use of cationicpolymers include their toxicity to cells and/or their lack of stability.

As such, there is a pressing need for new compounds, compositions, andmethods of using cationic components to improve delivery of therapeuticdrugs, including nucleic acids, to cells, tissues and organisms.

SUMMARY

One aspect of the description are compounds of formula I

wherein R₁ and R₂ is independently selected from a group consisting ofC₁₀ to C₁₈ alkyl, C₁₂ to C₁₈ alkenyl, and oleyl group; wherein R₃ and R₄are independently selected from a group consisting of C₁ to C₆ alkyl,and C₂ to C₆ alkanol; wherein X is selected from a group consisting of—CH₂—, —S—, and —O— or absent; wherein Y is selected from —(CH₂)_(n),—S(CH₂)_(n), —O(CH₂)_(n)—, thiophene, —SO₂(CH₂)_(n)—, and ester, whereinn=1-4; wherein a=1-4; wherein b=1-4; wherein c=1-4; and wherein Z is acounterion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts knockdown efficacy of certain embodiments of thedescription. This includes HEDC liposomes compared to DC-6-14 lipoplexcontrols.

FIG. 2 depicts an in vitro comparison of gene knockdown using cationiclipids.

FIG. 3 depicts an evaluation of gene expression in vivo with exemplaryHEDODC liposome formulations of the invention (* indicates p<0.05).

FIG. 4 depicts an evaluation of gene expression in vitro with exemplaryHEDC liposome formulations of the Example 15. Error bars indicatestandard deviations (n=3). A sigmoidal dose-response curve is shownbased on best fit. An EC₅₀ value was calculated from the curve. This isindicated to be 11.8 nM.

FIG. 5 shows the results of measurements in vivo using a rat DMNQ model.After subtracting background gp46 mRNA levels determined from the naïvegroup, all test group values were normalized to the average gp46 mRNA ofthe vehicle group (expressed as a percent of the vehicle group). Themean gp46 mRNA level following treatment showed dose-dependent responseand curve fitting to a sigmoidal dose response curve. The calculatedeffective dose, ED₅₀ value is 0.79 mg/kg.

FIG. 6 shows the results of measurements in vivo using a rat DMNC model.After subtracting background gp46 mRNA levels determined from the naïvegroup, all test group values were normalized to the average gp46 mRNA ofthe vehicle group (expressed as a percent of the vehicle group). MRPL19mRNA levels were determined by quantitative RT-PCR (TaqMan®) assay. mRNAlevels for gp46 were normalized to MRPL19 levels. (*** indicatesp<0.02.)

FIG. 7 shows the results of measurements in vivo using a rat pulmonarybleomycin model. The bar graph summarizes the fibrosis (T. Ashcroft)scoring of Azan-stained lung sections for each group. Statisticalanalysis was performed using a One way ANOVA, Bonferroni multicomparison test with Prism5 software.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Within the scope of the description are compounds of formula I

wherein R₁ and R₂ is independently selected from a group consisting ofC₁₀ to C₁₈ alkyl, C₁₂ to C₁₈ alkenyl, and oleyl group; wherein R₃ and R₄are independently selected from a group consisting of C₁ to C₆ alkyl,and C₂ to C₆ alkanol; wherein X is selected from a group consisting of—CH₂—, —S—, and —O— or absent; wherein Y is selected from —(CH₂)_(n),—S(CH₂)_(n), —O(CH₂)_(n)—, thiophene, —SO₂(CH₂)_(n)—, and ester, whereinn=1-4; wherein a=1-4; wherein b=1-4; wherein c=1-4; and wherein Z is acounterion.

Compounds of the invention are also referred to herein as being withinthe class of compounds known as “cationic lipids.” Cationic lipids arecompounds that include at least one lipid moiety and a positivelycharged nitrogen associated with a counterion. “Lipids” are understoodin the art to be comprised of a hydrophobic alkyl or alkenyl moiety anda carboxylic acid or ester moiety.

It has heretofore been discovered that the amino-alkyl-hydroxyl(—N-alkyl-OH) moiety of the compounds of formula I imparts properties tothe formulations of the invention not previously seen with othercationic lipids previously reported. Formulations of the invention thatinclude compounds of formula I result in superior reduction in proteinexpression, as compared to formulations that do not include compounds offormula I. Particularly surprising is the ability of formulations of theinvention that include compounds of formula I to reduce the expressionof HSP47.

Preferred compounds of the invention include those wherein R₁ and R₂ areeach independently C₁₀-C₃₀ alkyl. In more preferred embodiments, R₁ andR₂ are each independently C₁₀-C₂₀ alkyl. In even more preferredembodiments, R₁ and R₂ are each independently C₁₂-C₁₈ alkyl.Particularly preferred embodiments include those wherein R₁ and R₂ areeach independently C₁₃-C₁₇ alkyl. Most preferred are those compoundswherein R₁ and R₂ are each C₁₃ alkyl.

In other embodiments, R₁ and R₂ are each independently C₁₀-C₃₀ alkenyl.In more preferred embodiments, R₁ and R₂ are each independently C₁₀-C₂₀alkenyl. In still other embodiments, R₁ and R₂ are each independentlyC₁₂-C₁₈ alkenyl. In yet other embodiments, R₁ and R₂ are eachindependently C₁₃-C₁₇ alkenyl. Most preferred compounds of the inventioninclude those wherein R₁ and R₂ are each C₁₇ alkenyl.

Also for compounds of formula I, R₃ and R₄ are independently selectedfrom a group consisting of C₁ to C₆ alkyl. In preferred embodiments, R₃and R₄ are each independently C₁-C₃ alkyl. Most preferably, R₃ and R₄are each methyl. In other embodiments, at least one of R₃ and R₄ are—CH₂CH₂OH.

Most preferred are those compounds of formula I wherein a, b, and c areall 1.

Z can be any nitrogen counterion, as that term is readily understood inthe art. Preferred nitrogen counterions include halogens, with chlorideand bromide being particularly preferred and mesylate (SO₃CH₃ ⁻). Incontrast to other cationic lipids previously described wherein theeffect of the cationic lipid depends on the counterion, the efficacy ofcompounds of formula I, surprisingly, do not appear to be related to thecounterion selected.

Exemplary compounds of formula I include:

It is envisioned that any siRNA molecule can be used within the scope ofthe invention. the siRNA may include an antisense sequence to the mRNAcoding sequence for human hsp47 exemplified by SEQ ID NO:1, which isshown as follows.

ucuuuggcuu uuuuuggcgg agcuggggcg cccuccggaa 60 gcguuuccaa cuuuccagaaguuucucggg acgggcagga gggggugggg acugccauau 120 auagaucccg ggagcaggggagcgggcuaa gaguagaauc gugucgcggc ucgagagcga 180 gagucacguc ccggcgcuagcccagcccga cccaggccca ccguggugca cgcaaaccac 240 uuccuggcca ugcgcucccuccugcuucuc agcgccuucu gccuccugga ggcggcccug 300 gccgccgagg ugaagaaaccugcagccgca gcagcuccug gcacugcgga gaaguugagc 360 cccaaggcgg ccacgcuugccgagcgcagc gccggccugg ccuucagcuu guaccaggcc 420 auggccaagg accaggcaguggagaacauc cuggugucac ccgugguggu ggccucgucg 480 cuagggcucg ugucgcugggcggcaaggcg accacggcgu cgcaggccaa ggcagugcug 540 agcgccgagc agcugcgcgacgaggaggug cacgccggcc ugggcgagcu gcugcgcuca 600 cucagcaacu ccacggcgcgcaacgugacc uggaagcugg gcagccgacu guacggaccc 660 agcucaguga gcuucgcugaugacuucgug cgcagcagca agcagcacua caacugcgag 720 cacuccaaga ucaacuuccgcgacaagcgc agcgcgcugc aguccaucaa cgagugggcc 780 gcgcagacca ccgacggcaagcugcccgag gucaccaagg acguggagcg cacggacggc 840 gcccugcuag ucaacgccauguucuucaag ccacacuggg augagaaauu ccaccacaag 900 augguggaca accguggcuucauggugacu cgguccuaua ccgugggugu caugaugaug 960 caccggacag gccucuacaacuacuacgac gacgagaagg aaaagcugca aaucguggag 1020 augccccugg cccacaagcucuccagccuc aucauccuca ugccccauca cguggagccu 1080 cucgagcgcc uugaaaagcugcuaaccaaa gagcagcuga agaucuggau ggggaagaug 1140 cagaagaagg cuguugccaucuccuugccc aagggugugg uggaggugac ccaugaccug 1200 cagaaacacc uggcugggcugggccugacu gaggccauug acaagaacaa ggccgacuug 1260 ucacgcaugu caggcaagaaggaccuguac cuggccagcg uguuccacgc caccgccuuu 1320 gaguuggaca cagauggcaaccccuuugac caggacaucu acgggcgcga ggagcugcgc 1380 agccccaagc uguucuacgccgaccacccc uucaucuucc uagugcggga cacccaaagc 1440 ggcucccugc uauucauugggcgccugguc cggccuaagg gugacaagau gcgagacgag 1500 uuauagggcc ucagggugcacacaggaugg caggaggcau ccaaaggcuc cugagacaca 1560 ugggugcuau ugggguugggggggagguga gguaccagcc uuggauacuc cauggggugg 1620 ggguggaaaa acagaccgggguucccgugu gccugagcgg accuucccag cuagaauuca 1680 cuccacuugg acaugggccccagauaccau gaugcugagc ccggaaacuc cacauccugu 1740 gggaccuggg ccauagucauucugccugcc cugaaagucc cagaucaagc cugccucaau 1800 caguauucau auuuauagccagguaccuuc ucaccuguga gaccaaauug agcuaggggg 1860 gucagccagc ccucuucugacacuaaaaca ccucagcugc cuccccagcu cuaucccaac 1920 cucucccaac uauaaaacuaggugcugcag ccccugggac caggcacccc cagaaugacc 1980 uggccgcagu gaggcggauugagaaggagc ucccaggagg ggcuucuggg cagacucugg 2040 ucaagaagca ucgugucuggcguugugggg augaacuuuu uguuuuguuu cuuccuuuuu 2100 uaguucuuca aagauagggagggaaggggg aacaugagcc uuuguugcua ucaauccaag 2160 aacuuauuug uacauuuuuuuuuucaauaa aacuuuucca augacauuuu guuggagcgu 2208 ggaaaaaa

For example,

(SEQ. ID. NO. 2) Sense (5′->3′)GGACAGGCCUCUACAACUATT (SEQ. ID. NO. 3)Antisense(3′->5′)TTCCUGUCCGGAGAUGUUGAU.

Such compositions may also include an aqueous medium. Preferably, suchcompositions consist essentially of at least one compound of formula Iin a charge complex with the siRNA. Such compositions including acompound of formula I and an siRNA can further comprise a liquid medium.In one embodiment, the liquid medium is suitable for injection into aliving organism. Liquid mediums within the scope of any of the describedembodiments of the invention can be aqueous, that is, be comprisesentirely of an aqueous solvent, and to include salts, buffers, and/orother pharmaceutical excipients. In another embodiment, the liquidmedium may consist of an aqueous solvent in combination with anotherliquid solvent such as, for example, an organic solvent. Liquid mediumswithin the scope of any of the described embodiments of the inventioncan also include at least one organic solvent. Organic solvents areknown in the art per se and include C₁ to C₄ alcohols, dimethylsulfoxide (“DMSO”), and the like. Those liquid mediums that include amixture of water and at least one organic solvent are also within thescope of any of the described embodiments of the invention.

Also within the scope of the invention are compositions that comprise atleast one compound of formula I and a liposome. Some embodiments caninclude mixtures of compounds of formula I and a liposome. Otherembodiments can include a liposome and one or more compounds of formulaI in addition to cationic lipids that are not within the scope offormula I.

Compositions of the invention that include at least one compound offormula I and a liposome may further comprise one or more retinoidconjugates. In preferred embodiments of the invention, the retinoidconjugate will be present at a concentration of about 0.3 to about 30weight percent, based on the total weight of the composition orformulation, which is equivalent to about 0.1 to about 10 mol %, whichis equivalent to a molar ratio of about 0.1 to about 10. Preferably, theretinoid conjugate is a retinoid-linker-lipid molecule or aretinoid-linker-retinoid molecule.

An example of a retinoid conjugates include those compounds of formulaII:

wherein q, r, and s are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or10, and enantiomers and diastereomers thereof

Preferred compounds of formula II include those wherein q, r, and s areeach independently 1, 2, 3, 4, 5, 6, or 7. More preferred are thosecompounds of formula II wherein q, r, and s are each independently 3, 4,or 5. Most preferred are those compounds of formula II wherein q is 3, ris 5, and s is 3. One example of a compound of formula II is

DiVA-PEG-DiVA includes stereocenters and all enantiomers anddiastereomers are considered to be within the scope of the invention.

The concentration of cationic lipids in compositions of the invention,including those cationic lipids of formula I, can be from 1 to about 80weight percent, based on the total weight of the lipid composition. Morepreferably, the concentration is from 1 to about 75 weight percent. Evenmore preferably, the concentration is from about 30 to about 75 weightpercent. A concentration of from about 30 to about 75 weight percentcorresponds to about 30 to 60 mol. % and a molar ratio of about 30-60.Most preferred are those compositions having a cationic lipidconcentration of about 50 weight percent. In formulations that contain amixture of an ionizable cationic lipid and a quaternary amine cationiclipid of formula I, the preferred mol % is from 5% to 45 mol %, witheven more preferred mixture at approximately 20 mol % of the ionizablecationic lipid and 20 mol % of the quaternary amine cationic lipid forformula I.

Such compositions may also include an aqueous medium. The cationiclipids, including those of formula I, can be encapsulated within theliposome in such embodiments and may be inaccessible to the aqueousmedium. Furthermore, the cationic lipids, including those of formula I,can be localized on the outer surface of the liposome and be accessibleto the aqueous medium.

Compositions of the invention that include at least one compound offormula I and a liposome, and optionally a retinoid conjugate such as acompound of formula II, can also include siRNA.

In some embodiments, the siRNA will be encapsulated by the liposome sothat the siRNA is inaccessible to the aqueous medium. In otherembodiments, the siRNA will not be encapsulated by the liposome. In suchembodiments, the siRNA can be complexed on the outer surface of theliposome. In these embodiments, the siRNA is accessible to the aqueousmedium.

The forgoing compositions can also include PEG-conjugated lipids, whichare known in the art per se. Suitable PEG-lipids includePEG-phospholipids and PEG-ceramides such as, for example, PEG2000-DSPE,PEG2000-DPPE, PEG2000-DMPE, PEG2000-DOPE, PEG1000-DSPE, PEG1000-DPPE,PEG1000-DMPE, PEG1000-DOPE, PEGS 50-DSPE, PEG550-DPPE, PEG-550DMPE,PEG-1000DOPE, PEG-BML, PEG-Cholesterol. PEG2000-Ceramide,PEG1000-Ceramide, PEG750-Ceramide, PEG550-Ceramide.

For example, liposomes within the scope of the invention were preparedusing various PEG-lipids, incorporated using co-solubilization methodsdescribed herein. These formulations comprised cationiclipid:DOPE:cholesterol:DiVA-PEG-DiVA:PEG-Lipid (50:10:38:5:2 molarratio) and each formulation was tested in the pHSC in vitro assaydescribed herein using human/rat HSP-47-C siRNA at a concentration of200 nM. The results are shown in the following table:

gp46 Gene PEG-Lipid Knockdown (%) Std. Dev. Untreated 0 5.6 RNAimaxControl 50.0 7.9 PEG-BML 95.5 1.7 PEG1000-DMPE 93.2 0.8 PEG1000-DPPE92.8 1.3 PEG1000-DSPE 93.5 0.8 PEG1000-DOPE 90.7 2.5 PEG2000-Ceramide91.8 1.0 PEG2000-DMPE 93.7 3.4 PEG2000-DPPE 91.1 1.4 PEG2000-DSPE 89.41.7

The foregoing compositions of the invention can include one or morephospholipids such as, for example,1,2-distearoyl-sn-glycero-3-phosphocholine (“DSPC”),dipalmitoylphosphatidylcholine (“DPPC”),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (“DPPE”), and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (“DOPE”). Preferably, thehelper lipid is DOPE.

In addition to the cationic lipid of Formula I, other lipids may beuseful. These include ionizable cationic lipids, including 5104, shownbelow.

Delivery formulations may consist of a cationic lipid of Formula I incombination with an ionizable cationic lipid. An ionizable cationiclipid may include, e.g., 5104. The ionizable cationic lipid may bepresent at a concentration of 0 to 45 mol %, including a concentrationselected from 5, 10, 15, 20, 25, 30, 35 40, and 45 mol %.

Also within the scope of the invention are pharmaceutical formulationsthat include any of the aforementioned compositions in addition to apharmaceutically acceptable carrier or diluent. Pharmaceuticalformulations of the invention will include at least one therapeuticagent. Preferably, the therapeutic agent is an siRNA. It is envisionedthat any siRNA molecule can be used within the scope of the invention.

In preferred formulations of the invention including siRNA, the siRNA isencapsulated by the liposome. In other embodiments, the siRNA can beoutside of the liposome. In those embodiments, the siRNA can becomplexed to the outside of the liposome.

A useful range of cationic lipid:siRNA (lipid nitrogen to siRNAphosphate ratio, “N:P”) is 0.2 to 5.0. Particularly preferred range ofN:P is 1.5 to 2.5 for compositions and formulations of the invention.

Preferred formulations of the invention include those comprisingHEDC:S104:DOPE:cholesterol:PEG-DMPE:DiVA-PEG-DiVA (20:20:30:25:5:2 molarratio). Even more preferred embodiments include thoseHEDC:S104:DOPE:cholesterol:PEG-DMPE:DiVA-PEG-DiVA formulations whereinthe DiVA-PEG-DiVA is co-solubilized.

Other preferred formulations of the invention include those comprisingHE-DODC:S104:DOPE:cholesterol:PEG-DMPE:DiVA-PEG-DiVA (20:20:30:25:5:2molar ratio). Even more preferred embodiments include thoseHE-DODC:S104:DOPE:cholesterol:PEG-DMPE:DiVA-PEG-DiVA formulationswherein the DiVA-PEG-DiVA is co-solubilized.

Other preferred formulations of the invention include those comprisingDC-6-14:DOPE:cholesterol:DiVA-PEG-DiVA (40:30:30:5, molar ratios). Ineven more preferred embodiments, those formulations comprisingDC-6-14:DOPE:cholesterol:DiVA-PEG-DiVA include DiVA-PEG-DiVA that isco-solubilized.

Also within the scope of the invention are methods of delivering atherapeutic agent to a patient. These methods comprise providing apharmaceutical formulation including any of the foregoing compositionsand a pharmaceutically acceptable carrier or diluent; and administeringthe pharmaceutical formulation to the patient.

DEFINITIONS

As used herein, “alkyl” refers to a straight or branched fully saturated(no double or triple bonds) hydrocarbon group, for example, a grouphaving the general formula —C_(n)H_(2n+1). The alkyl group may have 1 to50 carbon atoms (whenever it appears herein, a numerical range such as“1 to 50” refers to each integer in the given range; e.g., “1 to 50carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2carbon atoms, 3 carbon atoms, etc., up to and including 50 carbon atoms,although the present definition also covers the occurrence of the term“alkyl” where no numerical range is designated). The alkyl group mayalso be a medium size alkyl having 1 to 30 carbon atoms. The alkyl groupcould also be a lower alkyl having 1 to 5 carbon atoms. The alkyl groupof the compounds may be designated as “C₁-C₄ alkyl” or similardesignations. By way of example only, “C₁-C₄ alkyl” indicates that thereare one to four carbon atoms in the alkyl chain, i.e., the alkyl chainis selected from the group consisting of methyl, ethyl, propyl,iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkylgroups include, but are in no way limited to, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like.

As used herein, “alkenyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more double bonds. Analkenyl group may be unsubstituted or substituted. When substituted, thesubstituent(s) may be selected from the same groups disclosed above withregard to alkyl group substitution unless otherwise indicated.

As used herein, “alkynyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more triple bonds. Analkynyl group may be unsubstituted or substituted. When substituted, thesubstituent(s) may be selected from the same groups disclosed above withregard to alkyl group substitution unless otherwise indicated.

As used herein, “halogen” refers to F, Cl, Br, and I.

As used herein, “mesylate” refers to —SO₃CH₃.

As used herein, the term “pharmaceutical formulation” refers to amixture of a composition disclosed herein with one or more otherchemical components, such as diluents or additional pharmaceuticalcarriers. The pharmaceutical formulation facilitates administration ofthe composition to an organism. Multiple techniques of administering apharmaceutical formulation exist in the art including, but not limitedto injection and parenteral administration.

As used herein, the term “pharmaceutical carrier” refers to a chemicalcompound that facilitates the incorporation of a compound into cells ortissues. For example dimethyl sulfoxide (DMSO) is a commonly utilizedcarrier as it facilitates the uptake of many organic compounds into thecells or tissues of an organism

As used herein, the term “diluent” refers to chemical compounds dilutedin water that will dissolve the formulation of interest (e.g., theformulation that can include a compound, a retinoid, a second lipid, astabilizing agent, and/or a therapeutic agent) as well as stabilize thebiologically active form of the formulation. Salts dissolved in bufferedsolutions are utilized as diluents in the art. One commonly usedbuffered solution is phosphate buffered saline because it mimics thesalt conditions of human blood. Since buffer salts can control the pH ofa solution at low concentrations, a buffered diluent rarely modifies thebiological activity of the formulation. As used herein, an “excipient”refers to an inert substance that is added to a formulation to provide,without limitation, bulk, consistency, stability, binding ability,lubrication, disintegrating ability, etc., to the composition. A“diluent” is a type of excipient.

As used herein, “therapeutic agent” refers to a compound that, uponadministration to a mammal in a therapeutically effective amount,provides a therapeutic benefit to the mammal. A therapeutic agent may bereferred to herein as a drug. Those skilled in the art will appreciatethat the term “therapeutic agent” is not limited to drugs that havereceived regulatory approval. A “therapeutic agent” can be operativelyassociated with a compound as described herein, a retinoid, and/or asecond lipid. For example, a second lipid as described herein can form aliposome, and the therapeutic agent can be operatively associated withthe liposome, e.g., as described herein.

As used herein, “lipoplex formulations” refer to those formulationswherein the siRNA is outside of the liposome. In preferred lipoplexformulations, the siRNA is complexed to the outside of the liposome.Other preferred lipoplex formulations include those wherein the siRNA isaccessible to any medium present outside of the liposome.

As used herein, “liposome formulations” refer to those formulationswherein the siRNA is encapsulated within the liposome. In preferredliposome formulations, the siRNA is inaccessible to any medium presentoutside of the liposome.

As used herein, the term “co-solubilized” refers to the addition of acomponent to the cationic lipid mixture before addition of an aqueousmedium and resulting formation of the vesicle.

As used herein, the term “decorated” refers to the addition of acomponent after vesicle formation in an aqueous solvent, in which thecomponent is preferentially localized on the surface of the vesicleaccessible to the aqueous medium outside of the vesicle.

As used herein, “DC-6-14” refers to the following cationic lipidcompound:

As used herein, “VA-PEG-VA” refers to the following compound:

VA-PEG-VA can be prepared according to methods known in the art. Apreferred method for prepared VA-PEG-VA is depicted in the followingscheme:

As used herein, a “retinoid” is a member of the class of compoundsconsisting of four isoprenoid units joined in a head-to-tail manner, seeG. P. Moss, “Biochemical Nomenclature and Related Documents,” 2nd Ed.Portland Press, pp. 247-251 (1992). “Vitamin A” is the genericdescriptor for retinoids exhibiting qualitatively the biologicalactivity of retinol. As used herein, retinoid refers to natural andsynthetic retinoids including first generation, second generation, andthird generation retinoids. Examples of naturally occurring retinoidsinclude, but are not limited to, (1) 11-cis-retinal, (2) all-transretinol, (3) retinyl palmitate, (4) all-trans retinoic acid, and (5)13-cis-retinoic acids. Furthermore, the term “retinoid” encompassesretinols, retinals, retinoic acids, rexinoids, and derivatives thereof.

As used herein, “retinoid conjugate” refers to a molecule that includesat least one retinoid moiety.

As used herein, “retinoid-linker-lipid molecule” refers to a moleculethat includes at least one retinoid moiety attached to at least onelipid moiety through at least one linker such as, for example, a PEGmoiety.

As used herein, “retinoid linker-retinoid molecule” refers to a moleculethat includes at least one retinoid moiety attached to at least oneother retinoid moiety (which may be the same or different) through atleast one linker such as, for example, a PEG moiety.

As used herein, the terms “lipid” and “lipophilic” are used herein intheir ordinary meanings as understood by those skilled in the art.Non-limiting examples of lipids and lipophilic groups include fattyacids, sterols, C₂-C₅₀ alkyl, C₂-C₅₀ heteroalkyl, C₂-C₅₀ alkenyl, C₂-C₅₀heteroalkenyl, C₅-C₅₀ aryl, C₅-C₅₀ heteroaryl, C₂-C₅₀ alkynyl, C₂-C₅₀heteroalkynyl, C₂-C₅₀ carboxyalkenyl, and C₂-C₅₀ carboxyheteroalkenyl. Afatty acid is a saturated or unsaturated long-chain monocarboxylic acidthat contains, for example, 12 to 24 carbon atoms A lipid ischaracterized as being essentially water insoluble, having a solubilityin water of less than about 0.01% (weight basis). As used herein, theterms “lipid moiety” and “lipophilic moiety” refers to a lipid orportion thereof that has become attached to another group. For example,a lipid group may become attached to another compound (e.g., a monomer)by a chemical reaction between a functional group (such as a carboxylicacid group) of the lipid and an appropriate functional group of amonomer.

As used herein, “siRNA” refers to small interfering RNA, also known inthe art as short interfering RNA or silencing RNA. siRNA is a class ofdouble stranded RNA molecules that have a variety of effects known inthe art, the most notable being the interference with the expression ofspecific genes and protein expression.

As used herein, “encapsulated by the liposome” refers to a componentbeing substantially or entirely within the liposome structure.

As used herein, “accessible to the aqueous medium” refers to a componentbeing able to be in contact with the aqueous medium.

As used herein, “inaccessible to the aqueous medium” refers to acomponent not being able to be in contact with the aqueous medium.

As used herein, “complexed on the outer surface of the liposome” refersto a component being operatively associated with the outer surface ofthe liposome in an aqueous solvent and accessible to aqueous mediumoutside of the liposome.

As used herein, “localized on the outer surface of the liposome” refersto a component being at or near the outer surface of the liposome in anaqueous solvent and accessible to aqueous medium outside of theliposome.

As used herein, “charge complexed” refers to an electrostaticassociation.

As used herein, the term “operatively associated” refers to anelectronic interaction between a compound as described herein, atherapeutic agent, a retinoid, and/or a second lipid. Such interactionmay take the form of a chemical bond, including, but not limited to, acovalent bond, a polar covalent bond, an ionic bond, an electrostaticassociation, a coordinate covalent bond, an aromatic bond, a hydrogenbond, a dipole, or a van der Waals interaction. Those of ordinary skillin the art understand that the relative strengths of such interactionsmay vary widely.

The term “liposome” is used herein in its ordinary meaning as understoodby those skilled in the art, and refers to a lipid bilayer structurethat contains lipids attached to polar, hydrophilic groups which form asubstantially closed structure in aqueous media. In some embodiments,the liposome can be operatively associated with one or more compounds,such as a therapeutic agent and a retinoid. A liposome may be comprisedof a single lipid bilayer (i.e., unilamellar) or it may comprised of twoor more lipid bilayers (i.e., multilamellar). While the interior of aliposome may consists of a variety of compounds, the exterior of theliposome is accessible to the aqueous formulation comprising theliposome. A liposome can be approximately spherical or ellipsoidal inshape.

Nucleic acid delivery systems may include, for example, aqueous andnonaqueous gels, creams, multiple emulsions, microemulsions, liposomes,ointments, aqueous and nonaqueous solutions, lotions, aerosols,hydrocarbon bases and powders, and can contain excipients such assolubilizers, permeation enhancers (e.g., fatty acids, fatty acidesters, fatty alcohols and amino acids), and hydrophilic polymers (e.g.,polycarbophil and polyvinylpyrolidone).

In addition to the cationic lipid of Formula I, other lipids may beuseful. These include ionizable cationic lipids, including

Delivery formulations may consist of a cationic lipid of Formula I incombination with an ionizable cationic lipid. An ionizable cationiclipid may include, e.g., 5104. The ionizable cationic lipid may bepresent at a concentration of 0 to 45 mol %, including a concentrationselected from 5, 10, 15, 20, 25, 30, 35, 40, and 45 mol %.

A lipid conjugated to a polyethylene glycol molecule (PEG), may bepresent in the liposome particles. PEG-lipids include

-   -   1,2-dimyristoleoyl-sn-glycero-3-phosphoethanolamine-N-PEG        (PEG-DMPE)    -   1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-PEG        (PEG-DPPE),    -   1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG        (PEG-DSPE), or    -   1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-PEG (PEG-DOPE)        and/or    -   PEG-ceramide.

Delivery formulations may consist of a cationic lipid of Formula I incombination with a PEG-lipid. The PEG-lipid may be present at aconcentration of 0 to 15 mol %, preferably 1 to 10 mol %, including aconcentration selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mole %.

Non-limiting examples of non-cationic lipids include phospholipids suchas lecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids arepreferably acyl groups derived from fatty acids having C₁₀-C₂₄ carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

In certain embodiments, the amount of phospholipid present in particlescomprises from about 0 mol % to about 55 mol %, more specifically at aconcentration selected from the 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,and 55%. As a non-limiting example, the phospholipid is DOPE.

Additional examples of non-cationic lipids include sterols such ascholesterol and derivatives thereof such as cholestanol, cholestanone,cholestenone, coprostanol, cholesteryl-2′-hydroxyethyl ether,cholesteryl-4′-hydroxybutyl ether, and mixtures thereof.

In certain embodiments, the cholesterol or cholesterol derivativepresent in particles comprises from about 0 mol % to about 55 mol %,more specifically at a concentration selected from the 5, 10, 15, 20,25, 30, 35, 40, 45, 50, and 55 mol %. As a non-limiting example,cholesterol is present in the lipid particles.

In certain other embodiments, the cholesterol present in lipid particlescontaining a mixture of phospholipid and cholesterol comprises fromabout 30 mol % to about 40 mol %, from about 30 mol % to about 35 mol %,or from about 35 mol % to about 40 mol % of the total lipid present inthe particle. As a non-limiting example, a lipid particle comprising amixture of phospholipid and cholesterol may comprise cholesterol atabout 34 mol % of the total lipid present in the particle.

In further embodiments, the cholesterol present in lipid particlescontaining a mixture of phospholipid and cholesterol comprises fromabout 10 mol % to about 30 mol %, from about 15 mol % to about 25 mol %,or from about 17 mol % to about 23 mol % of the total lipid present inthe particle. As a non-limiting example, a lipid particle comprising amixture of phospholipid and cholesterol may comprise cholesterol atabout 20 mol % of the total lipid present in the particle.

The retinoid or retinoid conjugate useful for delivery of nucleic acidis in a state in which it is dissolved in or mixed with a medium thatcan dissolve or retain it.

Any retinoid or retinoid conjugate may be used in the presentdescription as long as it is actively accumulated by stellate cells;examples of retinoid include, but are not limited to, tretinoin,adapalene, retinol palmitate, and in particular vitamin A, saturatedvitamin A, retinoic acid, and retinal. Examples of theretinoid-conjugate include PEG-retinoid conjugates. The presentdescription utilizes the property of stellate cells to positivelyincorporate a retinoid and/or a retinoid conjugate, and by using theretinoid and/or retinoid conjugate as a drug carrier or by bonding to orbeing included in another drug carrier component, a desired material orbody is transported specifically to stellate cells. A retinoid is amember of the class of compounds having a skeleton in which fourisoprenoid units are bonded in a head-to-tail manner. See G. P. Moss,“Biochemical Nomenclature and Related Documents,” 2nd Ed. PortlandPress, pp. 247-251 (1992). Vitamin A is a generic descriptor for aretinoid qualitatively showing the biological activity of retinol. Theretinoid in the present description promotes specific substance deliveryto a cancer cell and a CAF (that is, the substance is targeted at thesecells). Such a retinoid is not particularly limited, and examplesthereof include retinol, Vitamin A, saturated Vitamin A, retinal,retinoic acid, an ester of retinol and a fatty acid, an ester of analiphatic alcohol and retinoic acid, etretinate, tretinoin,isotretinoin, adapalene, acitretine, tazarotene, and retinol palmitate,and vitamin A analogues such as fenretinide, and bexarotene.Retinoid-conjugates include PEG-conjugates, e.g., diVA-PEG-diVA andVA-PEG-VA.

The drug carrier of the present description therefore may contain a drugcarrier component other than a retinoid and/or retinoid-conjugate. Sucha component is not particularly limited, and any component known in thefields of medicine and pharmacy may be used, but it is preferable for itto be capable of including a retinoid and/or retinoid conjugate.Furthermore, the drug carrier of the present description may contain asubstance that improves incorporation into stellate cells, for example,retinol-binding protein (RBP). The bonding or inclusion of the retinoidand/or retinoid conjugate with the drug carrier of the presentdescription may also be carried out by bonding or including the retinoidand/or retinoid conjugate with another component of the drug carrier bychemical and/or physical methods. Alternatively, bonding or inclusion ofthe retinoid and/or retinoid conjugate with the drug carrier of thepresent description may also be carried out by mixing the retinoidand/or retinoid conjugate having formation-affinity and basic componentsof the drug carrier, into the drug carrier components during preparationof the drug carrier.

The amount of retinoid and/or retinoid conjugate bonded to or includedin the drug carrier of the present description may be 0.01 mol % to 20mol % as a ratio by weight relative to the drug carrier components,preferably 0.2% to 10%, and more preferably 0.5% to 5 mol %, including aconcentration selected from the values 0.5, 1.0, 1.5, 2.0. 2.5, 3.0,3.5, 4.0, 4.5, and 5.0 mol %.

In certain embodiments, the present invention provides for a liposome tobe produced via mixing in a chamber. This includes a process thatprovides an aqueous solution comprising a nucleic acid such as aninterfering RNA in a first reservoir, providing an organic lipidsolution in a second reservoir, and mixing the aqueous solution with theorganic lipid solution such that the organic lipid solution mixes withthe aqueous solution so as to produce a liposome encapsulating thenucleic acid (e.g., siRNA) in a gradient of organic solventconcentration.

The liposome formed using the mixing method typically have a size offrom about 40 nm to about 250 nm, from about 50 nm to about 150 nm, fromabout 60 nm to about 150 nm. The particles thus formed do not aggregateand are optionally sized to achieve a uniform particle size.

The drug carrier of the present description may be in any form as longas a desired material or body can be transported to target stellatecells, and examples of the form include, but are not limited to, polymermicelle, liposome, emulsion, microsphere, and nanosphere. Furthermore,the drug carrier of the present description may include in its interiorthe substance that is to be transported, be attached to the exterior ofthe substance that is to be transported, or be mixed with the substancethat is to be transported as long as the retinoid and/or retinoidconjugate included therein is at least partially exposed on the exteriorof the preparation before it reaches the stellate cells at the latest.

The drug carrier of the present description specifically targetsstellate cells and enables a desired effect such as, for example,inhibition or prevention of fibrosis to be exhibited with the maximumeffect and minimum side effects by efficiently transporting to stellatecells a desired material or body such as, for example, a drug forcontrolling the activity or growth of stellate cells. The material orbody that the present drug carrier delivers is not particularly limited,but it preferably has a size that enables physical movement in a livingbody from an administration site to the liver, pancreas, etc., wherestellate cells are present. The drug carrier of the present descriptiontherefore can transport not only a material such as an atom, a molecule,a compound, a protein, or a nucleic acid but also a body such as avector, a virus particle, a cell, a drug release system constituted fromone or more elements, or a micromachine. The material or body preferablyhas the property of exerting some effect on stellate cells, and examplesthereof include one that labels stellate cells and one that controls theactivity or growth of stellate cells.

Therefore, in one embodiment of the present description, it is a drugfor controlling the activity or growth of stellate cells that the drugcarrier delivers. This may be any drug that directly or indirectlyinhibits the physicochemical actions of stellate cells involved in thepromotion of fibrosis, and examples thereof include, but are not limitedto, TGFβ activity inhibitors such as a truncated TGFβ type II receptorand a soluble TGFβ type II receptor, growth factor preparations such asHGF and expression vectors therefor, MMP production promoters such as anMMP gene-containing adenovirus vector, TIMP production inhibitors suchas an antisense TIMP nucleic acid, a PPARγ ligand, cell activationinhibitors and/or cell growth inhibitors such as an angiotensin activityinhibitor, a PDGF activity inhibitor, and a sodium channel inhibitor,and also apoptosis inducers such as compound 861 and gliotoxin,adiponectin, and a compound having Rho kinase inhibitory activity suchas (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane.Furthermore, the ‘drug for controlling the activity or growth ofstellate cells’ in the present description may be any drug that directlyor indirectly promotes the physicochemical actions of stellate cellsdirectly or indirectly involved in the inhibition of fibrosis, andexamples thereof include, but are not limited to, a drug for promoting acollagen degradation system, e.g., MMP production promoters such as anMMP expression vector, HGF, and drugs having HGF-like activity such asHGF analogues and expression vectors therefor.

Other examples of the drug for controlling the activity or growth ofstellate cells in the present description include a drug for controllingthe metabolism of an extracellular matrix such as collagen, for example,a substance having an effect in inhibiting the expression of a targetmolecule, such as siRNA, ribozyme, and antisense nucleic acid (includingRNA, DNA, PNA, and a composite thereof), a substance having a dominantnegative effect, and vectors expressing same, that target, for example,an extracellular matrix constituent molecule produced by stellate cellsor target one or more molecules that have the function of producing orsecreting the extracellular matrix constituent molecule.

The present description also relates to a medicine for treating astellate cell-related disorder, the medicine containing the drug carrierand the drug for controlling the activity or growth of stellate cells,and relates to the use of the drug carrier in the production of apharmaceutical composition for treating a stellate cell-relateddisorder. The stellate cell-related disorder referred to here means adisorder in which stellate cells are directly or indirectly involved inthe process of the disorder, that is, the onset, exacerbation,improvement, remission, cure, etc. of the disorder, and examples thereofinclude hepatic disorders such as hepatitis, in particular chronichepatitis, hepatic fibrosis, hepatic cirrhosis, and liver cancer, andpancreatic disorders such as pancreatitis, in particular chronicpancreatitis, pancreatic fibrosis, and pancreatic cancer.

In the medicine of the present description, the drug carrier may includea drug in its interior, be attached to the exterior of a drug-containingsubstance, or be mixed with a drug as long as the retinoid and/orretinoid-conjugate included in the drug carrier is at least partiallyexposed on the exterior of the preparation before it reaches thestellate cells at the latest. Therefore, depending on the route ofadministration or manner in which the drug is released, the medicine maybe covered with an appropriate material, such as, for example, anenteric coating or a material that disintegrates over time, or may beincorporated into an appropriate drug release system.

The present description therefore includes a drug carrier or medicinepreparation kit containing one or more containers containing one or moreof a drug carrier constituent, a retinoid and/or a retinoid conjugate,and/or a drug, and also includes an essential component for the drugcarrier or the medicine provided in the form of such a kit. The kit ofthe present description may contain, in addition to those describedabove, a description, etc. in which a preparation method or anadministration method for the drug carrier and the medicine of thepresent description is described. Furthermore, the kit of the presentdescription may contain all components for completing the drug carrieror the medicine of the present description but need not necessarilycontain all of the components. The kit of the present descriptiontherefore need not contain a reagent or a solvent that is normallyavailable at a place of medical treatment, an experimental facility,etc. such as, for example, sterile water, saline, or a glucose solution.

The present description further relates to a method for treating astellate cell-related disorder, the method including administering aneffective amount of the medicine to a subject in need thereof. Theeffective amount referred to here is an amount that suppresses onset ofthe target disorder, reduces symptoms thereof, or prevents progressionthereof, and is preferably an amount that prevents onset of the targetdisorder or cures the target disorder. It is also preferably an amountthat does not cause an adverse effect that exceeds the benefit fromadministration. Such an amount may be determined as appropriate by an invitro test using cultured cells, etc. or by a test in a model animalsuch as a mouse, a rat, a dog, or a pig, and such test methods are wellknown to a person skilled in the art.

In the method of the present description, the term ‘subject’ means anyliving individual, preferably an animal, more preferably a mammal, andyet more preferably a human individual. In the present description, thesubject may be healthy or affected with some disorder, and in the caseof treatment of a disorder being intended, the subject typically means asubject affected with the disorder or having a risk of being affected.

Furthermore, the term ‘treatment’ includes all types of medicallyacceptable prophylactic and/or therapeutic intervention for the purposeof the cure, temporary remission, prevention, etc. of a disorder. Forexample, when the disorder is hepatic fibrosis, the term ‘treatment’includes medically acceptable intervention for various purposesincluding delaying or halting the progression of fibrosis, regression ordisappearance of lesions, prevention of the onset of fibrosis, orprevention of recurrence.

The present description also relates to a method for delivering a drugto stellate cells using the drug carrier. This method includes, but isnot limited to, a step of supporting a substance to be delivered on thedrug carrier, and a step of administering or adding the drug carriercarrying the substance to be delivered to a stellate cell-containingliving body or medium, such as, for example, a culture medium. Thesesteps may be achieved as appropriate in accordance with any knownmethod, the method described in the present specification, etc. Thisdelivery method may be combined with another delivery method, forexample, another delivery method in which an organ where stellate cellsare present is the target, etc.

Nucleic acid molecules may be adapted for use to prevent or treatfibroses (e.g., liver, kidney, peritoneal, and pulmonary) diseases,traits, conditions and/or disorders, and/or any other trait, disease,disorder or condition that is related to or will respond to the levelsof hsp47 in a cell or tissue, alone or in combination with othertherapies. A nucleic acid molecule may include a delivery vehicle,including liposomes, for administration to a subject, carriers anddiluents and their salts, and/or can be present in pharmaceuticallyacceptable formulations.

The nucleic acid molecules of the description may include sequencesshown in Table 3. Examples of such nucleic acid molecules consistessentially of sequences provided in Table 3.

The nucleic acid molecules may be administered via pulmonary delivery,such as by inhalation of an aerosol or spray dried formulationadministered by an inhalation device or nebulizer, providing rapid localuptake of the nucleic acid molecules into relevant pulmonary tissues.Solid particulate compositions containing respirable dry particles ofmicronized nucleic acid compositions can be prepared by grinding driedor lyophilized nucleic acid compositions, and then passing themicronized composition through, for example, a 400 mesh screen to breakup or separate out large agglomerates. A solid particulate compositioncomprising the nucleic acid compositions of the description canoptionally contain a dispersant which serves to facilitate the formationof an aerosol as well as other therapeutic compounds. A suitabledispersant is lactose, which can be blended with the nucleic acidcompound in any suitable ratio, such as a 1 to 1 ratio by weight.

Aerosols of liquid particles may include a nucleic acid moleculesdisclosed herein and can be produced by any suitable means, such as witha nebulizer (see e.g., U.S. Pat. No. 4,501,729). Nebulizers arecommercially available devices which transform solutions or suspensionsof an active ingredient into a therapeutic aerosol mist either by meansof acceleration of a compressed gas, typically air or oxygen, through anarrow venturi orifice or by means of ultrasonic agitation. Suitableformulations for use in nebulizers include the active ingredient in aliquid carrier in an amount of up to 40% w/w preferably less than 20%w/w of the formulation. The carrier is typically water or a diluteaqueous alcoholic solution, preferably made isotonic with body fluids bythe addition of, e.g., sodium chloride or other suitable salts. Optionaladditives include preservatives if the formulation is not preparedsterile, e.g., methyl hydroxybenzoate, anti-oxidants, flavorings,volatile oils, buffering agents and emulsifiers and other formulationsurfactants. The aerosols of solid particles including the activecomposition and surfactant can likewise be produced with any solidparticulate aerosol generator. Aerosol generators for administeringsolid particulate therapeutics to a subject produce particles which arerespirable, as explained above, and generate a volume of aerosolcontaining a predetermined metered dose of a therapeutic composition ata rate suitable for human administration. One illustrative type of solidparticulate aerosol generator is an insufflator. Suitable formulationsfor administration by insufflation include finely comminuted powderswhich can be delivered by means of an insufflator. In the insufflator,the powder, e.g., a metered dose thereof effective to carry out thetreatments described herein, is contained in capsules or cartridges,typically made of gelatin or plastic, which are either pierced or openedin situ and the powder delivered by air drawn through the device uponinhalation or by means of a manually-operated pump. The powder employedin the insufflator consists either solely of the active ingredient or ofa powder blend comprising the active ingredient, a suitable powderdiluent, such as lactose, and an optional surfactant. The activeingredient typically includes from 0.1 to 100 w/w of the formulation. Asecond type of illustrative aerosol generator includes a metered doseinhaler. Metered dose inhalers are pressurized aerosol dispensers,typically containing a suspension or solution formulation of the activeingredient in a liquefied propellant. During use these devices dischargethe formulation through a valve adapted to deliver a metered volume toproduce a fine particle spray containing the active ingredient. Suitablepropellants include certain chlorofluorocarbon compounds, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane and mixtures thereof. The formulation canadditionally contain one or more co-solvents, for example, ethanol,emulsifiers and other formulation surfactants, such as oleic acid orsorbitan trioleate, anti-oxidants and suitable flavoring agents. Othermethods for pulmonary delivery are described in, e.g., US20040037780,U.S. Pat. No. 6,592,904, U.S. Pat. No. 6,582,728, and U.S. Pat. No.6,565,885. WO08132723 relates to aerosol delivery of oligonucleotides ingeneral, and of siRNA in particular, to the respiratory system.

Nucleic acid molecules may be administered to the central nervous system(CNS) or peripheral nervous system (PNS). Experiments have demonstratedthe efficient in vivo uptake of nucleic acids by neurons. See e.g.,Sommer et al., 1998, Antisense Nuc. Acid Drug Dev., 8:75; Epa et al.,2000, Antisense Nuc. Acid Drug Dev., 10:469; Broaddus et al., 1998, J.Neurosurg., 88:734; Karle et al., 1997, Eur. J. Pharmocol., 340:153;Bannai et al., 1998, Brain Research, 784:304; Rajakumar et al., 1997,Synapse, 26:199; Wu-pong et al., 1999, BioPharm, 12:32; Bannai et al.,1998, Brain Res. Protoc., 3:83; and Simantov et al., 1996, Neuroscience,74:39. Nucleic acid molecules are therefore amenable to delivery to anduptake by cells in the CNS and/or PNS.

Delivery of nucleic acid molecules to the CNS is provided by a varietyof different strategies. Traditional approaches to CNS delivery that canbe used include, but are not limited to, intrathecal andintracerebroventricular administration, implantation of catheters andpumps, direct injection or perfusion at the site of injury or lesion,injection into the brain arterial system, or by chemical or osmoticopening of the blood-brain barrier. Other approaches can include the useof various transport and carrier systems, for example though the use ofconjugates and biodegradable polymers. Furthermore, gene therapyapproaches, e.g., as described in Kaplitt et al., U.S. Pat. No.6,180,613 and Davidson, WO 04/013280, can be used to express nucleicacid molecules in the CNS.

Nucleic acid molecules may be formulated or complexed withpolyethylenimine (e.g., linear or branched PEI) and/or polyethyleniminederivatives, including for example grafted PEIs such as galactose PEI,cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI(PEG-PEI) derivatives thereof (see for example Ogris et al., 2001, AAPAPharmSci, 3, 1-11; Furgeson et al., 2003, Bioconjugate Chem., 14,840-847; Kunath et al., 2002, Pharm Res 19:810-17; Choi et al., 2001,Bull. Korean Chem. Soc., 22:46-52; Bettinger et al., 1999, BioconjugateChem., 10:558-561; Peterson et al., 2002, Bioconjugate Chem. 13:845-54;Erbacher et al., 1999, J Gene Med 1:1-18; Godbey et al., 1999., PNAS,96:5177-81; Godbey et al., 1999, J Controlled Release, 60:149-60;Diebold et al., 1999, J Biol Chem, 274:19087-94; Thomas et al., 2002,PNAS, 99, 14640-45; and Sagara, U.S. Pat. No. 6,586,524).

Nucleic acid molecules may include a bioconjugate, for example a nucleicacid conjugate as described in Vargeese et al., U.S. Ser. No.10/427,160; U.S. Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat.No. 6,235,886; U.S. Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S.Pat. No. 5,138,045.

Compositions, methods and kits disclosed herein may include anexpression vector that includes a nucleic acid sequence encoding atleast one nucleic acid molecule of the description in a manner thatallows expression of the nucleic acid molecule. Methods of introducingnucleic acid molecules or one or more vectors capable of expressing thestrands of dsRNA into the environment of the cell will depend on thetype of cell and the make up of its environment. The nucleic acidmolecule or the vector construct may be directly introduced into thecell (i.e., intracellularly); or introduced extracellularly into acavity, interstitial space, into the circulation of an organism,introduced orally, or may be introduced by bathing an organism or a cellin a solution containing dsRNA. The cell is preferably a mammalian cell;more preferably a human cell. The nucleic acid molecule of theexpression vector can include a sense region and an antisense region.The antisense region can include a sequence complementary to a RNA orDNA sequence encoding hsp47 and the sense region can include a sequencecomplementary to the antisense region. The nucleic acid molecule caninclude two distinct strands having complementary sense and antisenseregions. The nucleic acid molecule can include a single strand havingcomplementary sense and antisense regions.

Nucleic acid molecules that interact with target RNA molecules anddown-regulate gene encoding target RNA molecules (e.g., target RNAmolecules referred to by Genbank Accession numbers herein) may beexpressed from transcription units inserted into DNA or RNA vectors.Recombinant vectors can be DNA plasmids or viral vectors. Nucleic acidmolecule expressing viral vectors can be constructed based on, but notlimited to, adeno-associated virus, retrovirus, adenovirus, oralphavirus. The recombinant vectors capable of expressing the nucleicacid molecules can be delivered as described herein, and persist intarget cells. Alternatively, viral vectors can be used that provide fortransient expression of nucleic acid molecules. Such vectors can berepeatedly administered as necessary. Once expressed, the nucleic acidmolecules bind and down-regulate gene function or expression via RNAinterference (RNAi). Delivery of nucleic acid molecule expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from asubject followed by reintroduction into the subject, or by any othermeans that would allow for introduction into the desired target cell.

In another aspect, the present disclosure relates to a pharmaceuticalformulation comprising one or more physiologically acceptable surfaceactive agents, pharmaceutical carriers, diluents, excipients, andsuspension agents, or a combination thereof; and a formulation (e.g.,the formulation that can include a compound, a retinoid, a second lipid,a stabilizing agent, and/or a therapeutic agent) disclosed herein.Acceptable additional pharmaceutical carriers or diluents fortherapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, 18thEd., Mack Publishing Co., Easton, Pa. (1990), which is incorporatedherein by reference in its entirety. Preservatives, stabilizers, dyes,and the like may be provided in the pharmaceutical formulation. Forexample, sodium benzoate, ascorbic acid and esters of p-hydroxybenzoicacid may be added as preservatives. In addition, antioxidants andsuspending agents may be used. In various embodiments, alcohols, esters,sulfated aliphatic alcohols, and the like may be used as surface activeagents; sucrose, glucose, lactose, starch, crystallized cellulose,mannitol, light anhydrous silicate, magnesium aluminate, magnesiummetasilicate aluminate, synthetic aluminum silicate, calcium carbonate,sodium acid carbonate, calcium hydrogen phosphate, calcium carboxymethylcellulose, and the like may be used as excipients; coconut oil, oliveoil, sesame oil, peanut oil, soya may be used as suspension agents orlubricants; cellulose acetate phthalate as a derivative of acarbohydrate such as cellulose or sugar, or methylacetate-methacrylatecopolymer as a derivative of polyvinyl may be used as suspension agents;and plasticizers such as ester phthalates and the like may be used assuspension agents.

The pharmaceutical formulations described herein can be administered toa human patient per se, or in pharmaceutical formulations where they aremixed with other active ingredients, as in combination therapy, orsuitable pharmaceutical carriers or excipient(s). Techniques forformulation and administration of the compounds of the instantapplication may be found in “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., 18th edition, 1990.

Suitable routes of administration may include, for example, parenteraldelivery, including intramuscular, subcutaneous, intravenous,intramedullary injections, as well as intrathecal, directintraventricular, intraperitoneal, intranasal, or intraocularinjections. The formulation (e.g., the formulation that can include acompound, a retinoid, a second lipid, a stabilizing agent, and/or atherapeutic agent) can also be administered in sustained or controlledrelease dosage forms, including depot injections, osmotic pumps, and thelike, for prolonged and/or timed, pulsed administration at apredetermined rate. Additionally, the route of administration may belocal or systemic.

The pharmaceutical formulations may be manufactured in a manner that isitself known, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping or tabletting processes.

Pharmaceutical formulations may be formulated in any conventional mannerusing one or more physiologically acceptable pharmaceutical carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Proper formulation is dependent upon the route of administration chosen.Any of the well-known techniques, pharmaceutical carriers, andexcipients may be used as suitable and as understood in the art; e.g.,in Remington's Pharmaceutical Sciences, above.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, mannitol, lactose,lecithin, albumin, sodium glutamate, cysteine hydrochloride, and thelike. In addition, if desired, the injectable pharmaceuticalformulations may contain minor amounts of nontoxic auxiliary substances,such as wetting agents, pH buffering agents, and the like.Physiologically compatible buffers include, but are not limited to,Hanks's solution, Ringer's solution, or physiological saline buffer. Ifdesired, absorption enhancing preparations may be utilized.

Pharmaceutical formulations for parenteral administration, e.g., bybolus injection or continuous infusion, include aqueous solutions of theactive formulation (e.g., the formulation that can include a compound, aretinoid, a second lipid, a stabilizing agent, and/or a therapeuticagent) in water-soluble form. Additionally, suspensions of the activecompounds may be prepared as appropriate oily injection suspensions.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents that increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Theformulations may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

In addition to the preparations described previously, the formulationsmay also be formulated as a depot preparation. Such long actingformulations may be administered by intramuscular injection. Thus, forexample, the formulations (e.g., the formulation that can include acompound, a retinoid, a second lipid, a stabilizing agent, and/or atherapeutic agent) may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

The compositions and formulations of the invention may also beformulated for topical delivery and may be applied to the subject's skinusing any suitable process for application of topical delivery vehicle.For example, the formulation may be applied manually, using anapplicator, or by a process that involves both. Following application,the formulation may be worked into the subject's skin, e.g., by rubbing.Application may be performed multiple times daily or on a once-dailybasis. For example, the formulation may be applied to a subject's skinonce a day, twice a day, or multiple times a day, or may be applied onceevery two days, once every three days, or about once every week, onceevery two weeks, or once every several weeks.

Some embodiments herein are directed to a method of delivering atherapeutic agent to a cell. For example, some embodiments are directedto a method of delivering a therapeutic agent such as siRNA into a cell.Suitable cells for use according to the methods described herein includeprokaryotes, yeast, or higher eukaryotic cells, including plant andanimal cells (e.g., mammalian cells). In some embodiments, the cells canbe human fibrosarcoma cells (e.g., HT1080 cell line). In otherembodiments, the cells can be hepatic stellate cells (LX2 cell line). Inother embodiments, the cells can be cancer cells. In yet otherembodiments, the cells can be stem cells (pHSC cell line). Cell lineswhich are model systems for cancer may be used, including but notlimited to breast cancer (MCF-7, MDA-MB-438 cell lines), U87glioblastoma cell line, B16F0 cells (melanoma), HeLa cells (cervicalcancer), A549 cells (lung cancer), and rat tumor cell lines GH3 and 9 L.In these embodiments, the formulations described herein can be used totransfect a cell. These embodiments may include contacting the cell witha formulation described herein that includes a therapeutic agent, tothereby deliver a therapeutic agent to the cell.

Disclosed herein are methods for treating a condition characterized byabnormal fibrosis, which may include administering a therapeuticallyeffective amount of a formulation described herein. Conditionscharacterized by abnormal fibrosis may include cancer and/or a fibroticdisease. Types of cancer that may be treated or ameliorated by aformulation described herein include, but are not limited to, lungcancer, pancreatic cancer, breast cancer, liver cancer, stomach cancer,and colon cancer. In an embodiment, the cancer that may be treated orameliorated is pancreatic cancer. In another embodiment, the cancer thatmay be treated or ameliorated is lung cancer. Types of fibrotic diseasethat may be treated or ameliorated by a formulation described hereininclude, but are not limited to, hepatic fibrosis, hepatic cirrhosis,pancreatitis, pancreatic fibrosis, cystic fibrosis, vocal cord scarring,vocal cord mucosal fibrosis, laryngeal fibrosis, pulmonary fibrosis,idiopathic pulmonary fibrosis, cystic fibrosis, myelofibrosis,retroperitoneal fibrosis, and nephrogenic systemic fibrosis. In anembodiment, the condition that may be treated or ameliorated is hepaticfibrosis.

The formulations or pharmaceutical compositions described herein may beadministered to the subject by any suitable means. Non-limiting examplesof methods of administration include, among others, (a) administrationvia injection, subcutaneously, intraperitoneally, intravenously,intramuscularly, intradermally, intraorbitally, intracapsularly,intraspinally, intrasternally, or the like, including infusion pumpdelivery; (b) administration locally such as by injection directly inthe renal or cardiac area, e.g., by depot implantation; as well asdeemed appropriate by those of skill in the art for bringing the activecompound into contact with living tissue.

Pharmaceutical compositions suitable for administration includeformulations (e.g., the formulation that can include a compound, aretinoid, a second lipid, a stabilizing agent, and/or a therapeuticagent) where the active ingredients are contained in an amount effectiveto achieve its intended purpose. The therapeutically effective amount ofthe compounds disclosed herein required as a dose will depend on theroute of administration, the type of animal, including human, beingtreated, and the physical characteristics of the specific animal underconsideration. The dose can be tailored to achieve a desired effect, butwill depend on such factors as weight, diet, concurrent medication andother factors which those skilled in the medical arts will recognize.More specifically, a therapeutically effective amount means an amount ofcompound effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, canbe accomplished by one skilled in the art using routine pharmacologicalmethods. Typically, human clinical applications of products arecommenced at lower dosage levels, with dosage level being increaseduntil the desired effect is achieved. Alternatively, acceptable in vitrostudies can be used to establish useful doses and routes ofadministration of the compositions identified by the present methodsusing established pharmacological methods.

In non-human animal studies, applications of potential products arecommenced at higher dosage levels, with dosage being decreased until thedesired effect is no longer achieved or adverse side effects disappear.The dosage may range broadly, depending upon the desired effects and thetherapeutic indication. Typically, dosages may be about 10 microgram/kgto about 100 mg/kg body weight, preferably about 100 microgram/kg toabout 10 mg/kg body weight. Alternatively dosages may be based andcalculated upon the surface area of the patient, as understood by thoseof skill in the art.

The exact formulation, route of administration and dosage for thepharmaceutical compositions can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl et al. 1975, “ThePharmacological Basis of Therapeutics”, hereby incorporated herein inits entirety.) Typically, the dose range of the composition administeredto the patient can be from about 0.5 to about 1000 mg/kg of thepatient's body weight. The dosage may be a single one or a series of twoor more given in the course of one or more days, as is needed by thepatient. In instances where human dosages for compounds have beenestablished for at least some condition, the dosages will be about thesame, or dosages that are about 0.1% to about 500%, more preferablyabout 25% to about 250% of the established human dosage. Where no humandosage is established, as will be the case for newly-discoveredpharmaceutical compositions, a suitable human dosage can be inferredfrom ED₅₀ or ID₅₀ values, or other appropriate values derived from invitro or in vivo studies, as qualified by toxicity studies and efficacystudies in animals.

It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicityor organ dysfunctions. Conversely, the attending physician would alsoknow to adjust treatment to higher levels if the clinical response werenot adequate (precluding toxicity). The magnitude of an administrateddose in the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Although the exact dosage will be determined on a drug-by-drug basis, inmost cases, some generalizations regarding the dosage can be made. Thedaily dosage regimen for an adult human patient may be, for example, adose of about 0.1 mg to 2000 mg of each active ingredient, preferablyabout 1 mg to about 500 mg, e.g. 5 to 200 mg. In other embodiments, anintravenous, subcutaneous, or intramuscular dose of each activeingredient of about 0.01 mg to about 100 mg, preferably about 0.1 mg toabout 60 mg, e.g. about 1 to about 40 mg is used. In cases ofadministration of a pharmaceutically acceptable salt, dosages may becalculated as the free base. In some embodiments, the formulation isadministered 1 to 4 times per day. Alternatively the formulations may beadministered by continuous intravenous infusion, preferably at a dose ofeach active ingredient up to about 1000 mg per day. As will beunderstood by those of skill in the art, in certain situations it may benecessary to administer the formulations disclosed herein in amountsthat exceed, or even far exceed, the above-stated, preferred dosagerange in order to effectively and aggressively treat particularlyaggressive diseases or infections. In some embodiments, the formulationswill be administered for a period of continuous therapy, for example fora week or more, or for months or years.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themodulating effects, or minimal effective concentration (MEC). The MECwill vary for each compound but can be estimated from in vitro data.Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. However, HPLC assays orbioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compositionsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration.

The amount of formulation administered may be dependent on the subjectbeing treated, on the subject's weight, the severity of the affliction,the manner of administration and the judgment of the prescribingphysician.

Formulations disclosed herein (e.g., the formulation that can include acompound, a retinoid, a second lipid, a stabilizing agent, and/or atherapeutic agent) can be evaluated for efficacy and toxicity usingknown methods. For example, the toxicology of a particular compound, orof a subset of the compounds, sharing certain chemical moieties, may beestablished by determining in vitro toxicity towards a cell line, suchas a mammalian, and preferably human, cell line. The results of suchstudies are often predictive of toxicity in animals, such as mammals, ormore specifically, humans. Alternatively, the toxicity of particularcompounds in an animal model, such as mice, rats, rabbits, or monkeys,may be determined using known methods. The efficacy of a particularcompound may be established using several recognized methods, such as invitro methods, animal models, or human clinical trials. Recognized invitro models exist for nearly every class of condition, including butnot limited to cancer, cardiovascular disease, and various immunedysfunction. Similarly, acceptable animal models may be used toestablish efficacy of chemicals to treat such conditions. When selectinga model to determine efficacy, the skilled artisan can be guided by thestate of the art to choose an appropriate model, dose, and route ofadministration, and regime. Of course, human clinical trials can also beused to determine the efficacy of a compound in humans.

The formulations may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. The pack or dispensermay also be accompanied with a notice associated with the container inform prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals, which notice is reflective of approvalby the agency of the form of the drug for human or veterinaryadministration. Such notice, for example, may be the labeling approvedby the U.S. Food and Drug Administration for prescription drugs, or theapproved product insert. Compositions comprising a compound formulatedin a compatible pharmaceutical carrier may also be prepared, placed inan appropriate container, and labeled for treatment of an indicatedcondition.

It is understood that, in any compound described herein having one ormore stereocenters, if an absolute stereochemistry is not expresslyindicated, then each center may independently be of R-configuration orS-configuration or a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure or be stereoisomeric mixtures. Inaddition it is understood that, in any compound having one or moredouble bond(s) generating geometrical isomers that can be defined as Eor Z each double bond may independently be E or Z a mixture thereof.Likewise, all tautomeric forms are also intended to be included.

The invention can be further exemplified by reference to the followingexamples. These examples are illustrative, only, and are not intended tolimit the invention.

EXAMPLES Example 1 Preparation of2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethan-aminiumbromide (HEDC)

Preparation of Intermediate 1:2,2′-(tert-butoxycarbonylazanediyl)bis(ethane-2,1-diyl)ditetradecanoate(HEDC-BOC-IN)

A solution of N-BOC-diethanolamine (194 g, 0.946 mol), triethylamine(201 g, 2.03 mol) and diaminopyridine (23.1 g, 0.19 mol) in DCM (1750mL) was cooled to 0° C. A solution of myristoyl chloride (491 g, 1.99mol) in DCM (440 mL) was added over a period of 50 minutes at 0-10° C.,and the mixture was allowed to warm to ambient temperature. Fullconversion was indicated by TLC after 1.5 hours at 20-24° C. Water (1750mL) was added and pH 8.3 was measured by pH meter. The organic phase wasseparated, washed with (1) 6% NaHCO₃ (500 mL), (2) 0.3 M HCl (1700 mL),(3) 12.5% sodium chloride (1700 mL), and dried with anhydrous magnesiumsulphate (120 g). Evaporation of the filtrate at 50° C. and 50 mBar gave622 g of2,2′-(tert-butoxycarbonylazanediyl)bis(ethane-2,1-diyl)ditetradecanoate,(HEDC-BOC-IN). This distillation residue, which solidified on standing,was used in the next step.

Preparation of Intermediate 2:2,2′-(tert-butoxycarbonylazanediyl)bis(ethane-2,1-diyl)ditetradecanoate(HEDC-amine-IN) TFA salt

HEDC-BOC-IN (620 g, 0.990 mol) was transformed into a liquid by briefheating into a 50° C.-bath and then cooled below 25° C. TFA (940 mL,1.39 kg, 1.18 mol) was added to the liquid over a period of 30 minutes,using moderate cooling in order to maintain a temperature not higherthan 25° C. Having added two thirds of the amount of TFA, significantgas evolution was observed. The reaction mixture was stirred overnightat ambient temperature. TLC indicated traces of HEDC-BOC-IN. Thereaction mixture was heated for distillation of TFA under reducedpressure (125-60 mBar) from a water bath of 50-55° C., and distillationwas continued until it became very slow. TFA fumes were absorbed in ascrubber with 10% sodium hydroxide. Heptane (2000 mL) was added,stirred, and distilled off under reduced pressure. Heptane (2000 mL) wasadded to the partly solidified residue, and the mixture was heated to45° C., at which temperature a slightly turbid solution was formed. Thesolution was cooled, seeded at 40° C., and precipitate was formed bystirring for a period of 25 minutes at 40-36° C. Having cooled andstirred for a period of 40 minutes at ambient temperature, theprecipitate of heavy crystals was isolated by filtration, and the filtercake was washed with heptane (1000 mL). The wet filter cake (914 g) wasdried overnight at ambient temperature under reduced pressure (<1 mBar)to give 635 g (100%) of2,2′-(tert-butoxycarbonylazanediyl)bis(ethane-2,1-diyl)ditetradecanoate(HEDC-amine-IN) TFA salt as white crystals.

Preparation of Intermediate 3:2,2′42-(dimethylamino)acetylazanediyl)bis(ethane-2,1-diyl)ditetradeca-noate(HEDC-DiMeGly-IN)

N,N-Dimethylglycine (56.6 g, 548 mmol), HOBt hydrate (83.9 g, 548 mmol)and EDC hydrochloride (105 g, 548 mmol) were added to DMF (3.5 L), andthe mixture was stirred at ambient temperature for a period of one hour.A clear solution was formed. HEDC-amine-IN TFA salt (270 g, 442 mmol)was mixed with DCM (1.15 L) and 6% sodium bicarbonate (1.15 L). Theseparated organic phase with the free amine was added to the couplingmixture in DMF, and a precipitation as well as a temperature increase ofapprox. 9° C. was observed. Triethylamine (47.0 g, 464 mmol) was added,and the reaction mixture was stirred at 25-30° C. for a period of fivehours. TLC indicated incomplete conversion, and additional EDChydrochloride (29.5 g, 154 mmol) was added. Having stirred overnight atambient temperature, a clear solution was observed, and TLC nowindicated full conversion. The reaction mixture was mixed with DCM (2.3L) and 2% sodium bicarbonate (7 L). The organic phase was washed twicewith 1.25% sodium chloride (5 L each) and dried with anhydrous magnesiumsulphate (186 g). The filtrate was evaporated at 50° C. and 30 mBar togive 253 g of crude oil. The crude material was loaded to a columnpacked with 2.6 kg of Silica Gel 60 (40-63μ). The product was elutedwith toluene:ethyl acetate (8:2) (4 L), and followed with ethylacetate:methanol (1:1) (5 L). The product containing fractions (3.5-8 L)was evaporated (50° C./250-20 mBar) to give 208 g (66%) of2,2′-(2-(dimethylamino)acetylazanediyl)bis(ethane-2,1-diyl)ditetradecanoate(HEDC-DiMeGly-IN) as an oil.

Preparation of HEDC:2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxethanaminiumbromide

A mixture of HEDC-DiMeGly-IN (206 g, 337 mmol) and 2-bromoethanol (274g, 2.19 mol) was stirred at 80° C. for a period of two hours. HPLCindicated 8.1% of unreacted dimethylglycin-intermediate. After anadditional period of 40 minutes at 80° C., HPLC indicated 7.8% ofunreacted dimethylglycin intermediate. Warm (65° C.) ethyl acetate (2 L)was added. A blank filtration of the hot solution was washed with hotethyl acetate (0.5 L). The combined filtrates were cooled to 0° C., andcrystallization was initiated by seeding. The product suspension wascooled slowly and stirred at −16 to −18° C. for a period of 40 minutes.The precipitate was isolated by filtration, and the filter cake waswashed with cold ethyl acetate (200 mL). Drying overnight (20° C./<1mBar) gave 211 g of crude material. The material was then recrystallizedfrom a mixture of ethyl acetate (2.1 L) and ethanol (105 mL) by heatingto 35° C. and seeding at 25° C. The precipitate was isolated at 10° C.,washed with cold ethyl acetate (300 mL) and dried (20° C./<1 mBar)overnight to give 161 g (66%) of2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethanaminiumbromide (HEDC). HPLC indicated 99.5% purity. QTOF MS ESI+: m/z 655.6(M+H).

Example 2 Preparation of2-(bis(3-(tetradecanoyloxy)propyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxo-ethanaminiumbromide (Pr-HEDC)

Preparation of Intermediate 1: 3,3′-azanediylbis(propan-1-ol)

A mixture of 3-amino-1-propanol (14.5 mL, 19.0 mmol), 1-chloro-3-hydroxypropane (8 mL, 95.6 mmol) and H₂O (˜50 mL) was refluxed over 24 hours.Potassium hydroxide (5.40 g) was then added. After dissolution, thewhole of the H₂O was evaporated to leave viscous oil and largequantities of potassium chloride. These were filtered and washed withdry acetone and dichloromethane. The organic phase was dried overNa₂SO₄, filtered, and concentrated. Product was then purified via silicagel chromatography with a DCM/MeOH gradient to yield 12.5 g3,3′-azanediylbis(propan-1-ol).

Preparation of Intermediate 2: tert-butyl bis(3-hydroxypropyl)carbamate

3,3′-Azanediylbis(propan-1-ol) (12.5 g, 95.4 mmol) was diluted in DCM(25 mL). A solution of di-tert-butyl dicarbonate (26 g, 119.25 mmol) inDCM (25 mL) was slowly added while stirring under a blanket of Ar gas.Reaction was allowed to stir overnight. The reaction mixture wasconcentrated. Purification by silica gel chromatography with a DCM/MeOHgradient yielded tert-butyl bis(3-hydroxypropyl)carbamate.

Preparation of Intermediate 3:((tert-butoxycarbonyl)azanediyl)bis(propane-3,1-diyl)ditetradecanoate

tert-Butyl bis(3-hydroxypropyl)carbamate (4.00 g, 17.3 mmol),triethylamine (4.80 ml, 34.6 mmol) and 4-dimethylaminopyridine (529 mg,4.33 mmol) were dissolved in chloroform (50 mL). While being stirred inan ice-bath, a solution of myristoyl chloride was added in ˜15 min. Theaddition was carried out in such a way that the temperature of thereaction did not exceed 30° C. The reaction was allowed to stir at roomtemperature overnight. Next day, MeOH (50 mL) and 0.9% saline solution(50 mL) was added to quench the reaction. The organic layer wasseparated and washed with 1M NaHCO₃. Solvent was dried with Na₂SO₄,filtered, and concentrated in vacuo to yield((tert-butoxycarbonyl)azanediyl)bis(propane-3,1-diyl)ditetradecanoate asan oil.

Preparation of Intermediate 4:azanediylbis(propane-3,1-diyl)ditetradecanoate TFA salt

((tert-butoxycarbonyl)azanediyl)bis(propane-3,1-diyl)ditetradecanoate(11.3 g, 17.3 mmol) was dissolved in TFA/CHCl₃ (1:1, 20 mL) and themixture was allowed to stir at room temperature for 15 minutes. Themixture was then concentrated in vacuo. This was repeated a second time.Residue was then dissolved in DCM and washed with H₂O, dried withNa₂SO₄, and concentrated in vacuo. Purification by silica gelchromatography with a DCM/MeOH gradient yieldedazanediylbis(propane-3,1-diyl)ditetradecanoate as a TFA salt (750 mg).

Preparation of Intermediate 5:((2-(dimethylamino)acetyl)azanediyl)bis(propane-3,1-diyl)ditetradecanoate

Azanediylbis(propane-3,1-diyl)ditetradecanoate TFA salt (750 mg, 1.35mmol) was diluted with DCM (5 mL) and added to a pre-activated mixtureof N,N-dimethylglycine (154 mg, 1.49 mmol), HATU (616 mg, 1.62 mmol) andDIEA (495 uL, 2.84 mmol) in DCM (5 mL). Product was flushed with argonand stirred at room temperature overnight, and then concentrated.Purification by silica gel chromatography with a DCM/MeOH gradientyielded 465 mg((2-(dimethylamino)acetyl)azanediyl)bis(propane-3,1-diyl)ditetradecanoate.

Preparation of Pr-HEDC:2-(bis(3-(tetradecanoyloxy)propyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethanaminiumbromide

In a sealed system,((2-(dimethylamino)acetyl)azanediyl)bis(propane-3,1-diyl)ditetradecanoate(246 mg, 0.385 mmol) was dissolved in ACN (10 mL), and 2-bromoethanol(500 uL) was added. The reaction vessel was flushed with inert gas andthen sealed. The mixture was heated to 80° C., stirred overnight, andthen cooled and concentrated in vacuo. Purification by silica gelchromatography with a DCM/MeOH gradient yielded 99 mg2-(bis(3-(tetradecanoyloxy)propyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethanaminiumbromide. QTOF MS ESI+: m/z 683.6 (M+H).

Example 3 Preparation of2-(bis(3-(oleoyloxy)propyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethanaminiumbromide (Pr-HE-DODC)

Preparation of Intermediate 1:(Z)-((tert-butoxycarbonyl)azanediyl)bis(propane-3,1-diyl)dioleate

tert-Butyl bis(3-hydroxypropyl)carbamate (synthesis previouslydescribed), triethylamine and DMAP were dissolved in chloroform. Whilebeing stirred in an ice-bath, a solution of oleoyl chloride was added in15 min. The addition was carried out in such a way that the temperatureof the reaction did not exceed 30° C. The reaction was allowed to stirat room temperature overnight. Next day, MeOH (50 mL) and 0.9% salinesolution (50 mL) was added to quench the reaction. The organic layer wasseparated and washed with 1M NaHCO₃. Solvent was dried with Na₂SO₄,filtered and concentrated to yield an oil.(Z)-((tert-butoxycarbonyl)azanediyl)bis(propane-3,1-diyl)dioleate wascarried forward without and further purification.

Preparation of Intermediate 2:(Z)-azanediylbis(propane-3,1-diyl)dioleate TFA salt

(Z)-((tert-Butoxycarbonyl)azanediyl)bis(propane-3,1-diyl)dioleate (13.2g, 17.3 mmol) was dissolved in TFA/CHCl₃ (1:1, 20 mL) and the mixturewas allowed to stir at room temperature for 15 min. The mixture was thenconcentrated in vacuo. This was repeated a second time. Residue was thendissolved in DCM and washed with H₂O, dried with Na₂SO₄ andconcentrated. Purification by silica gel chromatography with a DCM/MeOHgradient yielded (Z)-azanediylbis(propane-3,1-diyl)dioleate TFA salt(750 mg).

Preparation of Intermediate 3:(Z)-((2-(dimethylamino)acetyl)azanediyl)bis(propane-3,1-diyl)dioleate

(Z)-azanediylbis(propane-3,1-diyl)dioleate TFA salt (750 mg, 1.13 mmol)was diluted with DCM (5 mL) and added to a pre-activated mixture ofN,N-dimethylglycine (128 mg, 1.24 mmol), HATU (517 mg, 1.36 mmol) andDIEA (413 uL, 2.37 mmol) in DCM (5 mL). Flask was flushed with argon andallowed to stir at room temperature overnight. The reaction mixture wasconcentrated, and subjected to silica gel chromatography with a DCM/MeOHgradient to yield(Z)-((2-(dimethylamino)acetyl)azanediyl)bis(propane-3,1-diyl)dioleate.

Preparation of Pr-HE-DODC:2-(bis(3-(oleoyloxy)propyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethanaminiumbromide

In a sealed system,(Z)-((2-(dimethylamino)acetyl)azanediyl)bis(propane-3,1-diyl)dioleate(269 mg, 0.360 mmol) was dissolved in ACN (10 mL) and 2-Bromoethanol(200 uL) was added. The reaction vessel was flushed with inert gas andthen sealed. Reaction was heated to 80° C. and allowed to stirovernight. The reaction mixture was cooled and concentrated.Purification by silica gel chromatography with a DCM/MeOH gradientyielded2-(bis(3-(oleoyloxy)propyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethanaminiumbromide (129 mg). QTOF MS ESI+: m/z 791.7 (M+H).

Example 4 Preparation of3-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-3-oxopro-pan-1-aminiumbromide (HE-Et-DC)

Preparation of Intermediate 1:((3-(dimethylamino)propanoyl)azanediyl)bis(ethane-2,1-diyl)ditetradeca-noate

Synthesis of azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA saltpreviously described. Azanediylbis(ethane-2,1-diyl)ditetradecanoate TFAsalt (1.5 g, 2.85 mmol) was diluted with DCM (10 mL) and added to apre-activated mixture of 3-(dimethylamino)propionic acid HCl salt (482mg, 3.14 mmol), HATU (1.30 g, 3.42 mmol) and DIEA (1.04 mL, 5.98 mmol)in DCM (10 mL). The round-bottomed flask was flushed with argon and thereaction mixture was allowed to stir at room temperature overnight. Thereaction mixture was concentrated. Purification by silica gelchromatography with a DCM/MeOH gradient yielded((3-(dimethylamino)propanoyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate.

Preparation of HE-Et-DC:3-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-3-oxopropan-1-aminiumbromide

In a sealed system,((3-(dimethylamino)propanoyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(606 mg, 0.970 mmol) was dissolved in ACN (10 mL) and 2-Bromoethanol(500 uL) was added. The reaction vessel was flushed with inert gas andthen sealed. Reaction was heated to 80° C. and stirred overnight, thencooled and concentrated. Purification by silica gel chromatography witha DCM/MeOH gradient yielded3-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-3-oxopropan-1-aminiumbromide (80 mg). QTOF MS ESI+: m/z 669.6 (M+H).

Example 5 Preparation of3-(bis(2-(oleoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-3-oxopropan-1-aminiumbromide (HE-Et-DODC)

Preparation of Intermediate 1:(Z)-((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)dioleate

N-Boc diethanolamine (Aldrich 15268, Lot#0001406013, MW 205.25; 17.81 g,0.087 mole), triethylamine (Aldrich, MW 101.19; 24.4 ml, 0.176 mole) and4-(dimethylamino)pyridine (Aldrich, MW 122.17; 2.76 g, 1.3 g, 0.023mole) were dissolved in 350 ml of chloroform. While being stirred, asolution of oleoyl chloride (MW 300.91; 61.6 g, 0.174 mole) in 100 ml ofchloroform was added in 10 min (Alternatively, the chloroform solutionof N-Boc diethanolamine was immersed in an ice/water bath while oleoylchloride was added). The addition was carried out in such a way that thetemperature of the reaction mixture does not exceed 50° C. The reactionmixture was stirred at room temperature for 2 hrs. A mixture of 200 mlof methanol (EMD MX0485-5, Lot 50350) and 200 ml of 0.9% saline (Sodiumchloride, BDH, BDH8014, Lot#92717) was added to quench the reaction. Theorganic layer was separated and was washed with 2×100 ml of diluteaqueous sodium bicarbonate (Aldrich S6014, Batch#095K0143). The solventwas removed by rotary evaporation to afford 59.5 g of crude product aspale yellow oil (MW 734.14; 59.5 g, 0.081 mole, 100% yield). Thismaterial was used for the next step without further purification. 1H NMR(400 MHz, CDCl3) 0.87 (t, 6H, CH3), 1.20-1.40 (m, 40H, CH2), 1.45 (s,9H, tBu CH3), 1.59 (m, 4H, CH2CH2C(═O)), 2.00 (m, 8H, CH2CH═CH), 2.33(t, 4H, CH2C(═O)), 3.48 (m, 4H, NCH2CH2O), 4.18 (m, 4H, NCH2CH2O), 5.33(m, 4H, CH═CH).

Preparation of Intermediate 2: (Z)-azanediylbis(ethane-2,1-diyl)dioleateTFA salt

The (Z)-((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)dioleate(59.5 g, 0.081 mole) was treated twice with 100 ml trifluoroacetic acid(Alfa Aesar Stock# A12198, Lot# D07W005, MW 114.02; 100 ml, 1.35 mole)and 100 ml of chloroform (Aldrich 154733, Lot# KBF5943V). Each consistedof stirring at room temperature for ten minutes, and the solvent wasremoved by rotary evaporation at the end of each treatment. After thesecond treatment, the reaction mixture was concentrated by rotaryevaporation. The residue was dissolved in 200 ml of methylene chlorideand the mixture had been washed with 100 ml of water twice. The residuewas purified by silica gel chromatography using a mixture of methanol(EMD MX0485-5, Lot 50350) and methylene chloride (EMD DX0835-5, Lot51090) as eluent to yield 44 g of(Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA salt (44.0 g). 1H NMR (400MHz, CDCl3) 0.87 (t, 6H, CH3), 1.20-1.40 (m, 40H, CH2), 1.59 (m, 4H,CH2CH2C(═O)), 2.00 (m, 8H, CH2CH═CH), 2.33 (t, 4H, CH2C(═O)), 3.31 (m,4H, NCH2CH2O), 4.38 (m, 4H, NCH2CH2O), 5.33 (m, 4H, CH═CH).

Preparation of Intermediate 3:(((Z)-((3-(dimethylamino)propanoyl)azanediyl)bis(ethane-2,1-diyl)dioleate

(Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA salt (1.50 g, 2.37 mmol)was diluted with DCM (10 mL) and added to a pre-activated mixture of3-(dimethylamino)propionic acid HCl salt (383 mg, 2.49 mmol), HATU (1034mg, 2.72 mmol) and DIEA (831 uL, 4.77 mmol) in DCM (10 mL). Theround-bottomed flask was flushed with argon and the reaction mixture wasallowed to stir at room temperature overnight. The reaction mixture wasconcentrated. Purification by silica gel chromatography with a DCM/MeOHgradient yielded(Z)-((3-(dimethylamino)propanoyl)azanediyl)bis(ethane-2,1-diyl)dioleate.

Preparation of HE-Et-DODC:3-(bis(2-(oleoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-3-oxo-propan-1-aminiumbromide

In a sealed system,(((Z)-((3-(dimethylamino)propanoyl)azanediyl)bis(ethane-2,1-diyl)dioleate(588 mg, 0.802 mmol) was dissolved in ACN (10 mL) and 2-bromoethanol(200 uL) was added. The reaction vessel was flushed with inert gas andthen sealed. Reaction was heated to 80° C. and stirred overnight, thencooled and concentrated in vacuo. Purification by silica gelchromatography with a DCM/MeOH gradient yielded3-(bis(2-(oleoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-3-oxopropan-1-aminiumbromide (160 mg). QTOF MS ESI+: m/z 764.3 (M+H).

Example 6 Preparation of4-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-4-oxobutan-1-aminiumbromide (HE-Pr-DC)

Preparation of Intermediate 1:((4-(dimethylamino)butanoyl)azanediyl)bis(ethane-2,1-diyl)ditetra-decanoate

Synthesis of azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA saltpreviously described. Azanediylbis(ethane-2,1-diyl)ditetradecanoate TFAsalt (1.00 g, 1.90 mmol) was diluted with DCM (5 mL) and added to apre-activated mixture of 4-(dimethylamino) butyric acid HCl salt (382mg, 2.28 mmol), HATU (867 mg, 2.28 mmol) and DIEA (728 uL, 4.18 mmol) inDCM (5 mL). The flask was flushed with argon and the reaction mixturewas stirred at room temperature overnight, then concentrated.Purification by silica gel chromatography with a DCM/MeOH gradientyielded((4-(dimethylamino)butanoyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate.

Preparation of HE-Pr-DC:4-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-4-oxobutan-1-aminiumbromide

In a sealed system,((4-(dimethylamino)butanoyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(300 mg, 0.469 mmol) was dissolved in ACN (5 mL) and 2-bromoethanol (500uL) was added. The reaction vessel was flushed with inert gas and thensealed. Reaction was heated to 80° C. and stirred overnight, thenconcentrated. Purification by silica gel chromatography with a DCM/MeOHgradient yielded4-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-4-oxobutan-1-aminiumbromide (140 mg). LCMS ESI+: m/z 684.4 (M+H).

Example 7 Preparation of4-(bis(2-(oleoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-4-oxobutan-1-aminiumbromide (HE-Pr-DODC)

Preparation of Intermediate 1:(Z)-((4-(dimethylamino)butanoyl)azanediyl)bis(ethane-2,1-diyl)dioleate

Synthesis of (Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA saltpreviously described. (Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA salt(1.00 g, 1.58 mmol) was diluted with DCM (5 mL) and added to apre-activated mixture of 4-(Dimethylamino) butyric acid HCl salt (317mg, 1.89 mmol), HATU (719 mg, 1.89 mmol) and DIEA (606 uL, 3.48 mmol) inDCM (5 mL). The flask was flushed with argon and the reaction mixturestirred at room temperature overnight, then concentrated. Purificationby silica gel chromatography with a DCM/MeOH gradient yielded(Z)-((4-(dimethylamino)butanoyl)azanediyl)bis(ethane-2,1-diyl)dioleate.

Preparation of HE-Pr-DODC:4-(bis(2-(oleoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-4-oxobutan-1-aminiumbromide

In a sealed system,((4-(dimethylamino)butanoyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(400 mg, 0.535 mmol) was dissolved in ACN (5 mL) and 2-Bromoethanol (500uL) was added. The reaction vessel was flushed with inert gas and thensealed. Reaction was heated to 80° C. and stirred overnight, thenconcentrated. Purification by silica gel chromatography with DCM/MeOHgradient yielded4-(bis(2-(oleoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-4-oxobutan-1-aminiumbromide (255 mg). LCMS ESI+: m/z 792.5 (M+H).

Example 8 Preparation of2-(bis(2-(oleoyloxy)ethyl)amino)-N,N-bis(2-hydroxyethyl)-N-methyl-2-oxoethanaminiumbromide (HE2DODC)

Preparation of Intermediate 1:(Z)-((2-bromoacetyl)azanediyl)bis(ethane-2,1-diyl)dioleate

Synthesis of (Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA saltpreviously described. (Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA salt(1.50 g, 2.34 mmol) was dissolved in DCM (20 mL) and placed in anice-bath. Bromoacetyl bromide (214 uL, 2.46 mmol) was added followed bytriethylamine (685 uL, 4.91 mmol). The ice-bath was removed and thereaction was allowed to stir overnight at room temperature under inertgas, then diluted with DCM to 100 mL, and washed with a 1M HCl (75 mL),H₂O (75 mL), saturated NaHCO₃ solution (75 mL) and saturated brinesolution (75 mL). All aqueous washes were back extracted with DCM (25mL). Dried organics with MgSO₄, filtered and concentrated in vacuo.Purification by silica gel chromatography with ethyl acetate yielded(Z)-((2-bromoacetyl)azanediyl)bis(ethane-2,1-diyl)dioleate (1.22 g).

Preparation of HE2DODC:2-(bis(2-(oleoyloxy)ethyl)amino)-N,N-bis(2-hydroxyethyl)-N-methyl-2-oxoethanaminiumbromide

In a sealed system,(Z)-((2-bromoacetyl)azanediyl)bis(ethane-2,1-diyl)dioleate (2.08 g, 2.75mmol) was combined with N-methyldiethylamine (1.58 mL, 13.8 mmol) wasadded. The reaction vessel was flushed with inert gas and then sealed.Reaction was heated to 50° C. and stirred overnight, and thenconcentrated. Purification by silica gel chromatography with DCM/MeOHgradient yielded2-(bis(2-(oleoyloxy)ethyl)amino)-N,N-bis(2-hydroxyethyl)-N-methyl-2-oxoethanaminiumbromide (479 mg). LCMS ESI+: m/z 793.7 (M+H).

Example 9 Preparation of2-(bis(2-((9Z,12Z)-octadeca-9,12-dienoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethanaminiumbromide (HEDC-DLin)

2-(bis(2-((9Z,12Z)-octadeca-9,12-dienoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethanaminiumbromide was prepared in similar fashion as HEDC with the substitution of(9Z,12Z)-octadeca-9,12-dienoyl chloride for myristoyl chloride.

Example 10 Preparation of2-(bis(2-(dodecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethanaminiumbromide (HEDC-12)

2-(bis(2-(Dodecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethanaminiumbromide was prepared in similar fashion as HEDC with the substitution ofdodecanoyl chloride for myristoyl chloride.

Example 11 Preparation of2-((2-(bis(2-(tetradecanoyloxy)ethyl)amino)-2-oxoethyl)thio)-N-(2-hydroxyethyl)-N,N-dimethylethanaminiumbromide (HES104)

Preparation of Intermediate 1:((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(S104)

Synthesis of azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA saltpreviously described. Azanediylbis(ethane-2,1-diyl)ditetradecanoate TFAsalt (152 g, 238 mmol) was stirred with DCM (2.3 L) and 10% potassiumbicarbonate (1.15 L) at 0-5° C. The organic phase was separated and theaqueous phase is further extracted with DCM (1.15 L). The combinedorganic phases were stirred with magnesium sulfate hydrate (236 g) for aperiod of 30 minutes at 0-5° C., filtrated and washed with DCM (1.15 L).To the combined filtrates were added2-((2-(dimethylamino)ethyl)thio)acetic acid hydrochloride (57.0 g, 285mmol), EDC hydrochloride (68.4 g, 357 mmol) and DMAP (2.91 g, 23.8mmol), and the suspension was stirred overnight at ambient temperature,after which period of time a clear solution was formed. MQ-water (2.3 L)and methanol (460 mL) were added and after having stirred for a periodof 10 minutes the clear organic phase was separated. The turbid aqueousphase (pH 3.0) was extracted with DCM (575 mL). The combined organicextracts were concentrated yielding 143 g of crude material as thehydrochloride salt. The crude material (142.6 g) was transferred to adistillation flask with DCM (500 mL), and ethyl acetate (1 L) was added.The solution was heated to distillation at atmospheric pressure, anddistillation was continued over a period of 70 minutes in order toobtain a temperature of the residue of 76° C. A total volume of 1.4 Lwas obtained by addition of ethyl acetate (800 mL), and ethanol (70 mL)was added. The clear solution at 50° C. was cooled to 37° C. and seedcrystals were added. Having observed initiation of significantcrystallization over a period of 10 minutes at 37-35° C., the suspensionwas cooled and stirred at 0° C. overnight and the precipitate wasisolated by filtration, and washed with cold ethyl acetate (210 mL).Drying to a constant weight at ambient temperature in oil pump vacuumover a period of 4.5 hours gave 134 g of recrystallized material as thehydrochloride salt, white crystalline solid. Tripotassium phosphate (85g, 0.40 mol) and dipotassium hydrogen phosphate (226 g, 1.30 mol) wasadded to purified water (1.7 L), and the solution formed with pH 10.9was cooled to 18-20° C. DCM (1.3 L) and recrystallized S104hydrochloride (133 g, 0.188 mol) were added, and the mixture was stirredfor a period of 10 min minutes. A clear organic phase was separated atmoderate rate (over a period of 35 minutes), and the turbid aqueousphase was further extracted with DCM (650 mL). The combined organicphases were stirred with anhydrous magnesium sulfate (65 g) for a periodof 40 min, and the mixture was filtered, washing with DCM (200 mL). Thecombined filtrates were evaporated from a 50° C. water bath underreduced pressure (down to 20 mBar, at which pressure evaporation wascontinued for one hour). Additional evaporation from a 15-20° C. waterbath at oil pump vacuum, resulted in 126 g partially solidified oil.Cooling in −20° C. cooling bath gave complete solidification, and afterdrying at −20° C. under an oil pump vacuum, we have obtained 126 g of((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(S104). HPLC indicated 98.1% purity.

Preparation of HES104:2-((2-(bis(2-(tetradecanoyloxy)ethyl)amino)-2-oxoethyl)thio)-N-(2-hydroxyethyl)-N,N-dimethylethanaminiumbromide

In a sealed system,((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(1.00 g, 1.49 mmol) was combined with 2-bromoethanol (687 uL, 9.69 mmol)was added. The reaction vessel was flushed with inert gas and thensealed. Reaction was heated to 75° C. and allowed to stir overnight,then cooled, and concentrated in vacuo. Purification by silica gelchromatography with DCM/MeOH gradient yielded2-((2-(bis(2-(tetradecanoyloxy)ethyl)amino)-2-oxoethyl)thio)-N-(2-hydroxyethyl)-N,N-dimethylethanaminiumbromide (HES104) (790 mg). LCMS ESI+: m/z 715.7 (M+H).

Example 12 Preparation of2-((2-(bis(2-(oleoyloxy)ethyl)amino)-2-oxoethyl)thio)-N-(2-hydroxyethyl)-N,N-dimethylethanaminiumbromide (HES104-DO)

Preparation of Intermediate 1:(Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)dioleate

Synthesis of (Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA saltpreviously described. (Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA salt(4.06 g, 6.41 mmol) was stirred in DCM (60 mL) with 10% K₂CO₃ (30 mL) at0-5° C. After 30 min, the organic phase was separated and the aqueousphase was further extracted with DCM (30 mL). The combined organicphases were stirred with anhydrous MgSO₄ for a period of 30 minutes at0-5° C., filtered, and washed with DCM (30 mL). To the combinedfiltrates were added to 2-((2-(dimethylamino)ethyl)thio)acetic acid(1.26 g, 7.70 mmol), EDC HCl salt (1.84 g, 9.62 mmol), DMAP (78.3 mg,0.64 mmol). The thin suspension was stirred overnight at roomtemperature; after which a period of time, the solution became clear.Next day, deionized water (60 mL) and methanol (30 mL) were added. Afterstirring for 10 minutes, the clear organic layer was isolated. Theturbid aqueous phase is extracted with DCM. The combined organicextracts were concentrated. Crude material was filtered through a plugof silica and taken up in DCM (40 mL) and PBS (pH=11, 50 mL) was added.The mixture was stirred at room temperature for 10 min. Then, theorganic phase was separated and the aqueous phase is extracted againwith DCM (15 mL). The combined organic phases were stirred withanhydrous MgSO₄ for a period of 30 min. The mixture was then filtered,and washed with DCM. The combined filtrates were concentrated in vacuoto yield(Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)dioleate(3.44 g).

Preparation of HES104-DO

In a sealed system,(Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)dioleate(540 mg, 0.693 mmol) was combined with 2-bromoethanol (319 uL, 4.50) wasadded. The reaction vessel was flushed with inert gas and then sealed.Reaction was heated to 75° C. and allowed to stir overnight. Next day,cooled and concentrated in vacuo. Purification by silica gelchromatography with DCM/MeOH gradient yielded2-((2-(bis(2-(oleoyloxy)ethyl)amino)-2-oxoethyl)thio)-N-(2-hydroxyethyl)-N,N-dimethylethanaminiumbromide (324 mg).

LCMS ESI+: m/z 823.8 (M+H).

Example 13 Preparation of2-((bis(2-(oleoyloxy)ethyl)carbamoyl)thio)-N-(2-hydroxyethyl)-N,N-dimethylethan-aminiumbromide (HETU104-DO)

Preparation of Intermediate 1:(Z)-((((2-(dimethylamino)ethyl)thio)carbonyl)azanediyl)bis(ethane-2,1-diyl)dioleate

Synthesis of(Z)-((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)dioleatepreviously described.(Z)-((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)dioleate (4.2 g,5.72 mmol) was dissolved in DCM (20 mL) and cooled to 0° C. in an icebath. TFA (20 mL) was added and the mixture was allowed to stir under ablanket of inert gas for 20 minutes. Afterwards, the mixture wasconcentrated in vacuo. The residue was partitioned between 10% K₂CO₃ (20mL) and DCM (20 mL). The mixture was stirred in an ice bath for 20minutes. Organic portion was collected, and turbid aqueous layer wasextracted with DCM (2×10 mL). The combined organic extracts were addedwith anhydrous MgSO₄ and stirred at 0° C. for 20 minutes. The suspensionwas filtered and washed DCM (10 mL). Diphosgene (1.38 mL, 11.4 mmol) wasadded to (Z)-azanediylbis(ethane-2,1-diyl)dioleate material in DCM andstirred under a blanket of inert gas at room temperature. Next day, DCMand excess diphosgene was removed in vacuo. 2-(dimethylamino)ethanethiol HCl salt (4.05 g, 28.6 mmol) was taken up in DCM (50 mL) andtriethylamine (5.2 mL, 37.2 mmol) and added to(Z)-((chlorocarbonyl)azanediyl)bis(ethane-2,1-diyl)dioleate residue.Reaction mixture was allowed to stir overnight at room temperature. Nextday, the mixture was diluted with DCM and washed with 0.3M HCl (75 mL),water (75 mL) and 10% K₂CO₃ (75 mL). Back extracted all aqueous washeswith DCM (25 mL). The organics was dried over anhydrous MgSO₄, filtered,and concentrated in vacuo. Purification by silica gel chromatographywith DCM/MeOH gradient yielded(Z)-((((2-(dimethylamino)ethyl)thio)carbonyl)azanediyl)bis(ethane-2,1-diyl)dioleate(1.90 g).

Preparation of HETU104DO

In a sealed system,(Z)-((((2-(dimethylamino)ethyl)thio)carbonyl)azanediyl)bis(ethane-2,1-diyl)dioleate(615 mg, 0.804 mmol) was combined with 2-Bromoethanol (370 uL, 5.22mmol) was added. The reaction vessel was flushed with inert gas and thensealed. Reaction was heated to 75° C. and allowed to stir overnight,then cooled and concentrated in vacuo. Purified by silica gelchromatography with a DCM/MeOH gradient yielded2-((bis(2-(oleoyloxy)ethyl)carbamoyl)thio)-N-(2-hydroxyethyl)-N,N-dimethylethanaminiumbromide (473 mg). LCMS ESI+: m/z 809.8 (M+H).

Example 14 Formation of Nucleic Acid-Lipid Particles

The siRNA referred to in the formulation protocols are double strandedsiRNA sequence with 21-mer targeting HSP47/gp46 wherein HSP47 (mouse)and gp46 (rat) are homologs—the same gene in different species:

rat HSP47-C double stranded siRNA used for in vitro assay (rat pHSCs)

(SEQ. ID NO. 2) Sense (5′->3′)GGACAGGCCUCUACAACUATT (SEQ. ID NO. 3)Antisense(3′->5′)TTCCUGUCCGGAGAUGUUGAUmouse HSP47-C double stranded siRNA used in formulations for in vivoassay (mouse CC14 model)

(SEQ. ID NO. 4) Sense (5′->3′)GGACAGGCCUGUACAACUATT (SEQ. ID NO. 5)Antisense(3′->5′)TTCCUGUCCGGACAUGUUGAU

Cationic Lipid Stock Preparation.

Stock solutions of cationic lipids were prepared by combining thecationic lipid with DOPE, cholesterol, and diVA-PEG-diVA in ethanol atconcentrations of 6.0, 5.1 and 2.7 and 2.4 mg/mL respectively. Ifneeded, solutions were warmed up to about 50° C. to facilitate thedissolution of the cationic lipids into solution.

Empty Liposome Preparation.

A cationic lipid stock solution was injected into a rapidly stirringaqueous mixture at 35-40° C. through injection needle(s) at 1.5 mL/minper injection port. The cationic lipid stock solution to the aqueoussolution ratio (v/v) is fixed at 35:65. Upon mixing, empty vesiclesformed spontaneously. The resulting vesicles were then allowed toequilibrate at 35-40° C. for 10 minutes before the ethanol content wasreduced to 12%. The empty liposomes were then diafiltered against 10×volumes of aqueous buffer to remove ethanol.

Lipoplex Preparation.

The empty vesicle prepared according to the above method was diluted tothe final volume of 1 mM concentration of cationic lipid by 9% sucrose.To the stirring solution, 100 μL of 5% glucose in RNase free water wasadded for every mL of the diluted empty vesicle (“EV”) and mixedthoroughly. 150 μL of 10 mg/mL siRNA solution in RNase free water wasthen added at once and mixed thoroughly. The mixture was then dilutedwith 5% glucose solution with 1.750 mL for every mL of the EV used. Themixture was stirred at about 200 rpm at room temperature for 10 minutes.Using a semi-permeable membrane with ˜100000 MWCO in a cross-flowultrafiltration system using appropriately chosen peristaltic pump (e.g. Midgee Hoop, UFP-100-H24LA), the mixture was concentrated to about ⅓of the original volume (or desired volume) and then diafiltered against5 times of the sample volume using an aqueous solution containing 3%sucrose and 2.9% glucose. The product was then filtered through acombined filter of 0.8/0.2 micron pore size under aseptic conditionsbefore use.

Formation of Non-diVA siRNA Containing Liposomes.

Cationic lipid, DOPE, cholesterol, and a PEG-conjugated lipid weresolubilized in absolute EtOH (200 proof) at a molar ratio of 50:10:38:2.The siRNA was solubilized in 50 mM citrate buffer and the temperaturewas adjusted to 35-40° C. The ethanol/lipid mixture was then added tothe siRNA-containing buffer while stirring to spontaneously form siRNAloaded liposomes. Lipids were combined with siRNA to reach a final totallipid to siRNA ratio of 5:1 to 15:1 (wt:wt). The siRNA loaded liposomeswere diafiltered against 10× volumes of PBS (pH 7.2) to remove ethanoland exchange the buffer. Final product was filtered through 0.22 μm,sterilizing grade, PES filter for bioburden reduction. This processyielded liposomes with a mean particle diameter of 50-100 nm, PDI<0.2,and >85% entrapment efficiency.

Formation of siRNA Containing Liposomes Co-Solubilized with diVA.

siRNA-diVA-Liposome formulations were prepared using the methoddescribed above. DiVA-PEG-diVA was co-solubilized in absolute ethanolwith the other lipids (cationic lipid, DOPE, cholesterol, andPEG-conjugated lipids at a ratio of 50:10:38:2) prior to addition to thesiRNA containing buffer. Molar content of diVA-PEG-diVA ranged from 0.1to 5 molar ratio. (50:10:38:2:0.1 to 50:10:38:2:5) This process yieldedliposomes with a mean particle diameter of 50-100 nm, PDI<0.2, and >85%entrapment efficiency.

Formation of siRNA Containing Liposomes with Cationic Lipids.

siRNA-diVA-Liposome formulations and siRNA-Liposome formulations wereprepared using the method described above. Cationic lipid can be, forexample, HEDC, HEDODC, DC-6-14, or any combination of these cationiclipids.

Formation of siRNA Containing Liposomes Decorated with diVA.

siRNA-Liposome formulations were prepared using the method describedabove and diluted to a siRNA concentration of 0.5 mg/mL in PBS. Cationiclipid can be HEDC, HEDODC, DC-6-14, or any combination of these cationiclipids. diVA-PEG-diVA was dissolved in absolute ethanol (200 proof) to afinal concentration ranging from 10 to 50 mg/mL. An appropriate amountof ethanol solution was added to the siRNA-Liposome solution to yield afinal molar percentage between 2 to 10 mol %. Solution was plunged upand down repeatedly with a pipette to mix. diVA-PEG-diVA concentrationand ethanol addition volume were adjusted to keep the additionvolume >1.0 μL and the final ethanol concentration <3% (vol/vol).Decorated liposomes were then incubated at ambient temperature for 1 hron an orbital shaker prior to in vitro or in vivo evaluation.

The following tables set forth preferred embodiments of the inventionexpressed in terms of molar ratios, as well as the equivalent mol % andweight percentages.

Molar Ratio Cationic PEG- Formulation Lipid DOPE Cholesterol lipid diVAHEDODC Liposome 50 10 38 2 — HEDODC Liposome + diVA 50 10 38 2 5 DC-6-14Lipoplex 40 30 30 — — DC-6-14 Lipoplex + diVA 40 30 30 — 5

Mol. % Cationic PEG- Formulation Lipid DOPE Cholesterol lipid diVAHE-DODC Liposome 50 10 38 2 — HE-DODC Liposome + 47.6 9.5 36.2 1.9 4.8diVA DC-6-14 Lipoplex 40 30 30 — — DC-6-14 Lipoplex + diVA 38.1 28.628.6 — 4.8

Weight Percent Cationic PEG- Formulation Lipid DOPE Cholesterol lipiddiVA HE-DODC Liposome 61.1 10.8 21.3 6.9 — HE-DODC Liposome + diVA 52.99.3 18.4 5.9 13.4 DC-6-14 Lipoplex 43.8 37 19.2 — — DC-6-14 Lipoplex +diVA 37.2 31.4 16.3 — 15.0

Example 15 Transfection with Liposomal Formulations

The transfection method is the same for LX-2 and pHSCs. The liposomeformulations or lipoplex formulations of the invention were mixed withgrowth medium at desired concentrations. 100 μl of the mixture was addedto the cells in 96-well plate and cells were incubated for 30 min at 37°C. in the incubator with 5% CO₂, After 30 min, medium was replaced withfresh growth medium. After 48 h of transfection, cells were processedusing Cell-to-Ct® lysis reagents (Applied Biosystems) according to themanufacturer's instructions.

Quantitative RT-PCR for Measuring HSP47 mRNA Expression (qRT-PCR)

HSP47 and GAPDH TaqMan assays and One-Step RT-PCR master mix werepurchased from Applied Biosystems. Each PCR reaction contained thefollowing composition: One-step RT-PCR mix 5 μl, TaqMan® RT enzyme mix0.25 μl, TaqMan® gene expression assay probe (HSP47) 0.25 μl, TaqMan®gene expression assay probe (GAPDH) 0.5 μl, RNase free water 3.25 μl,Cell lysate 0.75 μl, Total volume of 10 GAPDH was used as endogenouscontrol for the relative quantification of HSP47 mRNA levels.Quantitative RT-PCR was performed in ViiA 7 real-time PCR system(Applied Biosciences) using an in-built Relative Quantification method.All values were normalized to the average HSP47 expression of the mocktransfected cells and expressed as percentage of HSP47 expressioncompared to mock. In vivo experiments: Female C57B1/6 retired breedermice (Charles River) with a weight range of 24-30 grams were used forthis study. Animals were randomly distributed by weight into 10 groupsof 10 animals each. All animal procedures were approved by Bio-Quant'sIACUC and/or Attending Veterinarian as necessary and all animal welfareconcerns were addressed and documented. Mice were anesthetized withIsoflurane and exsanguinated via the inferior vena cava.

Up-regulation of heat shock protein 47 (HSP47) was induced viaintraperitoneal injecting CCl₄ (CCl₄ in olive oil, 1:7 (vol/vol), 1 μLper gram body weight) given every other day for 7 days (day 0, 2, 4, 6).On day 3 mice were treated for 4 consecutive days (day 3, 4, 5, 6) withliposome or lipoplex formulations of the invention or PBS by IVinjection into the tail vein. One group of ten mice (naïve) receivedneither CCl₄ treatment nor IV injection and served as the control groupfor normal HSP47 gene expression.

Experimental Timeline Day 0 1 2 3 4 5 6 7 CCl₄ IP Injection X X X X X XX Test Article IV Injection X X X X Sample Collection (n = 10) X

On day 7 and approximately 24 hours after final IV injection, allremaining mice were sacrificed and the livers were perfused with PBSprior to collecting liver samples for PCR analysis. An approximate 150mg sample from each mouse liver was collected and placed in 1.5 mLRNAlater stabilization reagent (Qiagen) and stored at 2-8° C. untilanalysis. Liver samples were not collected from areas of clear andmarked liver damage and/or necrosis.

Total RNA from mouse livers was extracted using RNeasy columns (Qiagen)according to the manufacturer's protocol. 20 ng of total RNA was usedfor quantitative RT-PCR for measuring HSP47 expression. HSP47 and GAPDHTaqMan® assays and One-Step RT-PCR master mix were purchased fromApplied Biosystems. Each PCR reaction contained the followingcomposition: One-step RT-PCR mix 5 μl, TaqMan® RT enzyme mix 0.25 μl,TaqMan® gene expression assay probe (HSP47) 0.25 μl, TaqMan® geneexpression assay probe (GAPDH) 0.5 μl, RNase free water 3.25 μl, RNA0.75 μl, Total volume of 10 μl. GAPDH was used as endogenous control forthe relative quantification of HSP47 mRNA levels. Quantitative RT-PCRwas performed in ViiA 7 realtime PCR system (Applied Biosciences) usingan in-built Relative Quantification method. All values were normalizedto the average HSP47 expression of the naïve animal group and expressedas percentage of HSP47 expression compared to naïve group.

The formulations described in FIG. 1 are as follows:

Molar Ratio Cationic Cho- PEG- VA or VA- Formulation Lipid DOPE lesterollipid conjugate DC-6-14 Lipoplex 40 30 30 — — DC-6-14 Lipoplex + VA* 4030 30 — 40 HEDC Liposome 50 10 38 2 — HEDC Liposome + VA- 50 10 38 2 5PEG-VA* HEDC Liposome + 50 10 38 2 5 diVA-PEG-diVA* *VA-PEG-VA anddiVA-PEG-diVA were added via co-solubilization. VA was added viadecoration post-process

The formulations described in FIG. 2 are as follows

Molar Ratio Cationic Cho- PEG- VA or VA- Formulation Lipid DOPE lesterollipid conjugate DC-6-14 Lipoplex 40 30 30 — — DC-6-14 Lipoplex + VA* 4030 30 — 40 DC-6-14 Liposome 50 10 38 2 DC-6-14 Liposome + 50 10 38 2 5diVA-PEG-diVA* HEDODC Liposome 50 10 38 2 — HEDODC Liposome + 50 10 38 25 diva-PEG-diVA* *diVA-PEG-diVA was added via co-solubilization. VA wasadded via decoration post-process.

The formulations described in FIG. 3 are as follows.

Molar Ratio Cationic Cho- PEG- VA or VA- Formulation Lipid DOPE lesterollipid conjugate DC-6-14 Lipoplex 40 30 30 — — DC-6-14 Lipoplex + VA* 4030 30 — 40 HEDODC Liposome 50 10 38 2 — HEDODC Liposome + 50 10 38 2 5diVA-PEG-diVA* *diVA-PEG-diVA was added via co-solubilization. VA wasadded via decoration post-process

Example 16 In Vitro Efficacy (pHSC), Dose Response

pHSC In Vitro Assay Description:

Primary hepatic stellate cells (pHSCs) in a 96-well plate were incubatedwith formulations (HEDC:S104:DOPE:Chol:Peg-DMPE:DiVA, 20:20:30:25:5:2)of increasing siRNA concentration. After 30 minutes, cells were washedand treated with fresh growth medium and incubated at 37° C. for 48hours. At that time, cells were lysed and gp46 and GAPDH mRNA levelswere measured by quantitative RT-PCR (TaqMan®) assay. mRNA levels ofgp46 were normalized to GAPDH levels. Normalized gp46 levels areexpressed as the percent of untreated control cells. FIG. 4 shows theresults. Error bars indicate standard deviations (n=3). Fitting data toa sigmoidal dose-response curve using Graphpad yielded an EC₅₀ of 11.8nM.

Example 17 Toxicity

HepG2 Cytotoxicity Assay Description

HepG2 cells, an adherent cell line derived from human hepatocellularcarcinoma, was cultured in MEM/EBSS (Hyclone, Logan, Utah, Cat#SH30024.01) supplemented with 10% FBS (Hyclone, Logan, Utah Cat#SH30910). HepG2 cells were seeded in 96-well Optilux black plates (BDFalcon, Cat # BD353220) at the density of 5000 cells/well overnight.Formulations were added to each well to final indicated siRNAconcentration (n=3). At 48 h post formulation addition, cell viabilitywas determined using CellTiter-Glo Luminescent Cell Viability Assay Kit(Promega, Cat #G7572) following manufacture's instruction.Chemiluminescent signal were measured on Clarity Luminescence MicroplateReader (502-Biotek, Winooski, Vt.). Viability was calculated based on %of chemiluminescent signal in formulation treated well normalizedagainst mock treated wells.

Combinations of quaternary amine cationic lipids of formula I with theirrespective ionizable synthetic precursors were evaluated (as shown inthe examples below, i-DC and HEDC, INT4 and DODC, 5104 and HES104).

The following table provides exemplary results from differentformulations. Combinations of quaternary amine cationic lipids withionizable cationic lipids surprisingly and unexpectedly were less toxicthan liposomes containing a single cationic lipid (see examples HEDC vs.HEDC+iDC; and DODC vs. DODC+INT4 in table below). The HEDC+S104combination was identified as another preferred formulation.

in vitro tox (% in vitro cell viability, Variant KD* HepG2 DescriptionFormulation Variants (%) @ 200 nM) Cationic Lipid 40 mol % HEDC, no 90%@ 27% Content (2 mol % ionizable lipid 200 nM PEG-Lipid) 50 mol % DODC,no 90% @ 55% ionizable lipid 200 nM 25 mol % DODC:25 mol % 90% @ 90%INT4 200 nM 20 mol % HEDC:20 mol % 89% @ 57% i-DC 200 nM 20 mol %HEDC:20 mol % 90% @ 52% S104 200 nM 10 mol % HEDC:30 mol % 90% @ 71%S104 200 nM  5 mol % HEDC:35 mol % 90% @ 80% S104 200 nM PEG Lipid  2mol % 70% Content  5 mol % 60% (DMPE-PEG)  7 mol % 55% 10 mol % 45% DIVAContent 0.25 mol %   35% (w/5 mol % 0.5 mol %  40% DMPE-PEG) 1.0 mol % 60% 2.0 mol %  70% DOPE:Cholesterol  DOPE-0%:Cholesterol 55% 89% Ratio(w/5 mol %  DOPE-5%:Cholesterol 50% 82% DMPE-PEG) DOPE-10%:Cholesterol45% 77% DOPE-15%:Cholesterol 40% 74% DOPE-20%:Cholesterol 35% 80%DOPE-25%:Cholesterol 30% 79% DOPE-30%:Cholesterol 25% 82%DOPE-35%:Cholesterol 20% 80% DOPE-40%:Cholesterol 15% 84%DOPE-45%:Cholesterol 10% 80% DOPE-50%:Cholesterol 5%  78%DOPE-55%:Cholesterol 0%  72% siRNA:Total Lipid 0.07 80% Ratio (w/5 mol %0.09 75% DMPE-PEG) 0.11 82% *All data with 20 mol % HEDC, 20 mol % S104,and 2 mol % DiVA @ 50 nM siRNa dose unless otherwise notedIn Vivo Toxicity

The HEDC:S104 (20:20) formulation is exceptionally well tolerated inpreliminary in vivo toxicity studies. No toxicity is observed when theformulation is injected intravenously at doses up to 25 mg/kg (rat) and12 mg/kg (monkey). This is considered to be superior in this field. Forexample, Alnylam Pharmaceuticals on Mar. 1, 2012 disclosed at theAsiaTIDES conference that their least toxic third generation lipidnanoparticles had a NOAEL of 10 mg/kg (single dose, rat).

Example 18 In Vivo Efficacy (Rat DMNQ)

In vivo activity of target formulation was evaluated in the short-termliver damage model (referred to as the Quick Model, DMNQ). In thismodel, the short-term liver damage induced by treatment with ahepatotoxic agent such as dimethylnitrosamine (DMN) is accompanied bythe elevation of gp46 mRNA levels. To induce these changes, maleSprague-Dawley rats were injected intraperitoneally with DMN on sixconsecutive days. At the end of the DMN treatment period, the animalswere randomized to groups based upon individual animal body weight.Formulations were administered as a single intravenous dose, one hourafter the last injection of DMN. Twenty four hours after, liver lobeswere excised and both gp46 and MRPL19 mRNA levels were determined byquantitative RT-PCR (TaqMan®) assay. Levels of gp46 mRNA were normalizedto MRPL19 levels.

Male Sprague-Dawley rats were treated with DMN at 10 mg/kg on day 1, 2,3 and 5 mg/kg on day 4, 5, 6 through intraperitoneally dosing to induceliver damage. Animals (n=8/group) were injected intravenously eitherwith formulations at a dose of 0.5, 0.75, 1.0, 2 mg/kg siRNA in aformulation consisting of HEDC:S104:DOPE:Chol:Peg-DMPE:DiVA(20:20:30:25:5:2), or PBS (vehicle), one hour after the last injectionof DMN. Twenty four hours later, total siRNA was purified from a sectionof the right liver lobe from each animal and stored at 4° C. until RNAisolation. Control groups included a PBS vehicle group (DMN-treated) andnaïve (untreated; no DMN) group. FIG. 5 shows the results ofmeasurements. After subtracting background gp46 mRNA levels determinedfrom the naïve group, all test group values were normalized to theaverage gp46 mRNA of the vehicle group (expressed as a percent of thevehicle group). The mean gp46 mRNA level following treatment showeddose-dependent response and curve fitting to sigmoidal dose responsecurve yielded EC₅₀ of 0.79 mg/kg.

Example 19 In Vivo Efficacy (Rat DMNC)

Male Sprague Dawley rats (130-160 g) were treated DMN throughintraperitoneally dosing to induce liver fibrosis. The DMN treatmentregimen was 3 times each week (Mon, Wed, and Fri) with 10 mg/kg (i.e.,5.0 mg/mL of DMN at a dose of 2.0 mL/kg body weight) for first 3 weeksand half dose of 5 mg/kg (i.e., 5 mg/mL of DMN at a dose of 1.0 mL/kg)from day 22 to 57. The sham group animals were injected with PBS(solvent for DMN) using the same schedule. On day 22, 24 h post the lastDMN treatment, blood samples were collected and assayed for liverdisease biomarkers to confirm the effectiveness of the DMN treatment.DMN treated animals were assigned to different treatment groups based onbody weight to ensure that the mean body weights and the range of bodyweights of the animals in each group have no significant difference.Animals from pretreatment group were sacrificed on day 25 to evaluatethe disease progression stage prior to treatment begins. Treatments withformulations containing gp46 siRNA were started at day 25 with twotreatments/week at specified siRNA dose for a total of 10 times. On day59, 48 hours after last formulation treatment and 72 hours after lastDMN treatment, animals were sacrificed by CO₂ inhalation. Liver lobeswere excised and both gp46 and MRPL19 mRNA levels were determined byquantitative RT-PCR (TaqMan®) assay. mRNA levels for gp46 werenormalized to MRPL19 levels.

Male Sprague-Dawley rats were treated with DMN at 10 mg/kg for threeweeks (three times/week) and then 5 mg/kg from day 22 to 57 (threetimes/week) through intraperitoneal dosing to induce liver fibrosis.Animals (n=ten/group) were injected intravenously either withformulations consisting of HEDC:S104:DOPE:Chol:Peg-DMPE:DiVA(20:20:30:25:5:2) at 1.5, 1.0, 0.75, and 0.5 mg/kg siRNA or PBS(vehicle) for 10 times (2 times/week), one hour after the last injectionof DMN. At day 59, total siRNA was purified from a section of the rightliver lobe from each animal and stored at 4° C. until RNA analysis.Control groups included a PBS vehicle group (DMN-induced, PBS treated,n=7) and sham group (PBS treated in place of DMN and formulation, n=10)group. FIG. 6 shows the results of measurements. After subtractingbackground gp46 mRNA levels determined from the naïve group, all testgroup values were normalized to the average gp46 mRNA of the vehiclegroup (expressed as a percent of the vehicle group). Animal frompretreat group (n=7) were sacrificed on day 25 to evaluate diseaseprogression level prior to treatment began. One-way Anova analysisfollowed by Dunnett's test showed significant gp46 gene knockdown in alltreatment groups as compared to vehicle group (***, P<0.001).

The following table summarizes the compounds described herein, and theresults obtained by testing these compounds in vivo and in vitro.

i. in vitro (pHSC) % KD ii. in vivo (rat Cationic DMNQ) Lipid NameStructure % KD Pr-HEDC

i. 75% @ 50 nM Pr-HE-DODC

i. 73% @ 50 nM HE-Et-DC

i. 70% @ 50 nM HE-Et-DODC

i. 71% @ 50 nM HE-Pr-DC

i. 47% @ 50 nM HE-Pr-DODC

i. 75% @ 50 nM HE2DODC

i. 78% @ 50 nM HEDC-DLin

i. 50% @ 50 nM HEDC

i. 68% @ 50 nM ii. 52% @ 0.5 mpk HEDC-12

i. 0% @ 50 nM HES104

HES104DO

HETU104DO

Example 20 In Vivo Anti-Pulmonary-Fibrosis

Male S-D rats (8 rats/group, 8 weeks old, Charles River LaboratoriesJapan, Inc.) were administered once with 0.45 mg bleomycin (BLM)dissolved in 0.1 mL of saline into the lung by intratracheallycannulating (MicroSprayer, Penn-Century, Inc.) under anesthesia, toproduce a bleomycin pulmonary fibrosis model. With this method, asignificant fibrosis occurs in the lung generally after approximately 2weeks. The Liposome formulation (1.5 mg/kg as an amount of siRNA, 1ml/kg in volume, i.e., 200 μl for a rat of 200 g) or PBS (1 ml/kg involume) was administered to the rats via the tail vein, starting fromthe 2 weeks after the bleomycin administration, for total of ten times(every other day). The rats were sacrificed at two days post lasttreatment, histological investigation of the lung tissue was performed(see FIG. 7). One way ANOVA and Bonferroni multi comparison test wasused for the evaluation of statistically-significant difference.

A part of the removed lung was formalin-fixed in accordance with aroutine method, and subjected to azan staining (azocarmine, aniline blueorange G solution).

As shown by the results of histological scoring (T. Ashcroft score) inFIG. 7, in the formulation administration group (Treatment), fibrosisscore was significantly decreased.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

Applicants reserve the right to physically incorporate into thisapplication any and all materials and information from any sucharticles, patents, patent applications, or other physical and electronicdocuments.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the description disclosedherein without departing from the scope and spirit of the description.Thus, such additional embodiments are within the scope of the presentdescription and the following claims. The present description teachesone skilled in the art to test various combinations and/or substitutionsof chemical modifications described herein toward generating nucleicacid constructs with improved activity for mediating RNAi activity. Suchimproved activity can include improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying nucleic acidmolecules with improved RNAi activity.

The descriptions illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “a” and “an” and “the” and similar referents in the context ofdescribing the description (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.The terms “comprising”, “having,” “including,” containing”, etc. shallbe read expansively and without limitation (e.g., meaning “including,but not limited to,”). Recitation of ranges of values herein are merelyintended to serve as a shorthand method of referring individually toeach separate value falling within the range, unless otherwise indicatedherein, and each separate value is incorporated into the specificationas if it were individually recited herein. All methods described hereincan be performed in any suitable order unless otherwise indicated hereinor otherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the description and does not pose alimitation on the scope of the description unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the description.Additionally, the terms and expressions employed herein have been usedas terms of description and not of limitation, and there is no intentionin the use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe description claimed. Thus, it should be understood that although thepresent description has been specifically disclosed by preferredembodiments and optional features, modification and variation of thedescriptions embodied therein herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this description.

The description has been described broadly and generically herein. Eachof the narrower species and subgeneric groupings falling within thegeneric disclosure also form part of the description. This includes thegeneric description of the description with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.Other embodiments are within the following claims. In addition, wherefeatures or aspects of the description are described in terms of Markushgroups, those skilled in the art will recognize that the description isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

What is claimed:
 1. A stellate-cell-specific drug carrier comprising astellate cell specific amount of a retinoid molecule and a liposomalcomposition wherein the liposomal composition comprises a cationic lipidof formula I

wherein R₁ and R₂ are independently selected from the group consistingof C₁₀ to C₁₈ alkyl, C₁₂ to C₁₈ alkenyl, and oleyl; wherein R₃ and R₄are independently selected from the group consisting of C₁ to C₆ alkyl,and C₂ to C₆ alkanol; wherein X is selected from the group consisting of—CH₂—, —S—, and —O—, or is absent; wherein Y is selected from—(CH₂)_(n)—, —S(CH₂)_(n)—, —O(CH₂)_(n)—, -thiophene-, —SO₂(CH₂)_(n), andester, wherein n=1-4; wherein a=1-4; wherein b=1-4; wherein c=1-4; andwherein Z is a counterion.
 2. The drug carrier of claim 1, wherein theliposomal composition comprises a lipid vesicle comprising a bilayer oflipid molecules.
 3. The drug carrier of claim 1, wherein the retinoidmolecule is a compound of formula II:

wherein q, r, and s are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or10; or an enantiomer, diastereomer, or mixture of stereoisomers thereof.4. The drug carrier of claim 3, wherein the retinoid molecule is


5. The drug carrier of claim 2, wherein the retinoid molecule is atleast partially exposed on the exterior of the drug carrier before thedrug carrier reaches the stellate cell.
 6. The drug carrier of claim 1,wherein the retinoid molecule is 0.1 mol % to 20 mol % of the lipidmolecules.
 7. The drug carrier of claim 1, wherein the cationic lipid isat a concentration of 5 mol % to 50 mol %.
 8. The drug carrier of claim2, wherein cationic lipid is


9. The drug carrier of claim 2, wherein the lipid molecules furthercomprise a PEG-lipid.
 10. The drug carrier of claim 9, wherein thePEG-lipid is at a concentration of 1 to 10 mol %.
 11. The drug carrierof claim 9, wherein the PEG-lipid ispolyethyleneglycol-dimyristoyl-phosphatidylethanolamine.
 12. The drugcarrier of claim 2, wherein the lipid molecules further comprise anionizable cationic lipid.
 13. The drug carrier of claim 12, wherein theionizable cationic lipid is


14. The drug carrier of claim 12, wherein the ionizable cationic lipidis present at a concentration of 5 to 45 mol %.
 15. The drug carrier ofclaim 2, wherein the lipid molecules further comprise a combination of aquaternary amine cationic lipid and an ionizable cationic lipid.
 16. Thedrug carrier of claim 15, wherein the ionizable cationic lipid is theimmediate synthetic precursor to the quaternary amine cationic lipid.17. The drug carrier of claim 15, wherein the quaternary amine cationiclipid is HEDC and the ionizable cationic lipid is S104.
 18. The drugcarrier of claim 2, wherein the lipid molecules further comprise anon-cationic lipid.
 19. The drug carrier of claim 18, wherein thenon-cationic lipid is a phospholipid.
 20. The drug carrier of claim 19,wherein the phospholipid is1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.
 21. The drug carrier ofclaim 19, wherein the phospholipid is at a concentration of 0 to 55 mol%.
 22. The drug carrier of claim 18, wherein the non-cationic lipid ischolesterol.
 23. The drug carrier of claim 22, wherein cholesterol is ata concentration of 0 to 55 mol %.
 24. The drug carrier of claim 1,further comprising a nucleic acid.
 25. The drug carrier of claim 24,wherein the nucleic acid is an siRNA that is capable of knocking downexpression of hsp47 mRNA in the stellate cell.
 26. The drug carrier ofclaim 1, further comprising siRNA.
 27. The drug carrier of claim 26,wherein the siRNA is encapsulated by the liposome and is inaccessible toan aqueous medium.
 28. The drug carrier of claim 26, wherein the siRNAis complexed on the outer surface of the liposome and accessible to anaqueous medium.
 29. The drug carrier of claim 1, wherein the cationiclipid is selected from the group consisting of